Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi.

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state.

It was shown decades ago that purified 30S ribosome subunits readily interconvert between "active" and "inactive" conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactive-to-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch.

DNABII proteins play a central role in UPEC biofilm structure.

Most chronic and recurrent bacterial infections involve a biofilm component, the foundation of which is the extracellular polymeric substance (EPS). Extracellular DNA (eDNA) is a conserved and key component of the EPS of pathogenic biofilms. The DNABII protein family includes integration host factor (IHF) and histone-like protein (HU); both are present in the extracellular milieu. We have shown previously that the DNABII proteins are often found in association with eDNA and are critical for the structural integrity of bacterial communities that utilize eDNA as a matrix component. Here, we demonstrate that uropathogenic Escherichia coli (UPEC) strain UTI89 incorporates eDNA within its biofilm matrix and that the DNABII proteins are not only important for biofilm growth, but are limiting; exogenous addition of these proteins promotes biofilm formation that is dependent on eDNA. In addition, we show that both subunits of IHF, yet only one subunit of HU (HupB), are critical for UPEC biofilm development. We discuss the roles of these proteins in context of the UPEC EPS.

Distinct functional classes of ram mutations in 16S rRNA.

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.

Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state.

After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge--namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu-GTP-aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.

Evaluation of the kinetics and mechanism of action of anti-integration host factor-mediated disruption of bacterial biofilms.

The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice-like network wherein a member of the DNABII family of DNA-binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using non-typeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF-specific and mediated by binding of transiently dissociated IHF by anti-IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA-binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.

Role of helix 44 of 16S rRNA in the fidelity of translation initiation.

The molecular mechanisms that govern translation initiation to ensure accuracy remain unclear. Here, we provide evidence that the subunit-joining step of initiation is controlled in part by a conformational change in the 1408 region of helix h44. First, chemical probing of 30S initiation complexes formed with either a cognate (AUG) or near-cognate (AUC) start codon shows that an IF1-dependent enhancement at A1408 is reduced in the presence of AUG. This change in reactivity is due to a conformational change rather than loss of IF1, because other portions of the IF1 footprint are unchanged and high concentrations of IF1 fail to diminish the reactivity difference seen at A1408. Second, mutations in h44 such as A1413C stimulate 50S docking and cause reduced reactivity at A1408. Third, streptomycin, which has been shown by Rodnina and coworkers to stimulate 50S docking by reversing the inhibitory effects of IF1, also causes reduced reactivity at A1408. Collectively, these data support a model in which IF1 alters the A1408 region of h44 in a way that makes 50S docking unfavorable, and canonical codon-anticodon pairing in the P site restores h44 to a docking-favorable conformation. We also find that, in the absence of factors, the cognate 30S•AUG•fMet-tRNA ternary complex is >1000-fold more stable than the near-cognate 30S•AUC•fMet-tRNA complex. Hence, the selectivity of ternary complex formation is inherently high, exceeding that of initiation in vivo by more than 10-fold.

Noninflammatory 97-amino acid High Mobility Group Box 1 derived polypeptide disrupts and prevents diverse biofilms.

BACKGROUND: Bacterial biofilm communities are embedded in a protective extracellular matrix comprised of various components, with its' integrity largely owed to a 3-dimensional lattice of extracellular DNA (eDNA) interconnected by Holliday Junction (HJ)-like structures and stabilised by the ubiquitous eubacterial DNABII family of DNA-binding architectural proteins. We recently showed that the host innate immune effector High Mobility Group Box 1 (HMGB1) protein possesses extracellular anti-biofilm activity by destabilising these HJ-like structures, resulting in release of biofilm-resident bacteria into a vulnerable state. Herein, we showed that HMGB1's anti-biofilm activity was completely contained within a contiguous 97 amino acid region that retained DNA-binding activity, called 'mB Box-97'. METHODS: We engineered a synthetic version of this 97-mer and introduced a single amino acid change which lacked any post-translational modifications, and tested its activity independently and in combination with a humanised monoclonal antibody that disrupts biofilms by the distinct mechanism of DNABII protein sequestration. FINDINGS: mB Box-97 disrupted and prevented biofilms, including those formed by the ESKAPEE pathogens, and importantly reduced measurable proinflammatory activity normally associated with HMGB1 in a murine lung infection model. INTERPRETATION: Herein, we discuss the value of targeting the ubiquitous eDNA-dependent matrix of biofilms via mB Box-97 used singly or in a dual host-augmenting/pathogen-targeted cocktail to resolve bacterial biofilm infections. FUNDING: This work was supported by NIH/NIDCD R01DC011818 to L.O.B. and S.D.G. and NIH/NIAID R01AI155501 to S.D.G.

Short spacing between the Shine-Dalgarno sequence and P codon destabilizes codon-anticodon pairing in the P site to promote +1 programmed frameshifting.

Programmed frameshifting in the RF2 gene (prfB) involves an intragenic Shine-Dalgarno (SD) sequence. To investigate the role of SD-ASD pairing in the mechanism of frameshifting, we have analysed the effect of spacing between the SD sequence and P codon on P-site tRNA binding and RF2-dependent termination. When the spacing between an extended SD sequence and the P codon is decreased from 4 to 1 nucleotide (nt), the dissociation rate (k(off) ) for P-site tRNA increases by > 100-fold. Toeprinting analysis shows that pre-translocation complexes cannot be formed when the spacer sequence is ≤ 2 nt. Instead, the tRNA added secondarily to fill the A site and its corresponding codon move spontaneously into the P site, resulting in a complex with a 3 nt longer spacer between the SD-ASD helix and the P codon. While close proximity of the SD clearly destabilizes P-site tRNA, RF2-dependent termination and EF-Tu-dependent decoding are largely unaffected in analogous complexes. These data support a model in which formation of the SD-ASD helix in ribosomes stalled at the in-frame UGA codon of prfB generates tension on the mRNA that destabilizes codon-anticodon pairing in the P site and promotes slippage of the mRNA in the 5' direction.

A role for the 30S subunit E site in maintenance of the translational reading frame.

The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the beta-hairpin of ribosomal protein S7. This mutation (S7DeltaR77-Y84) increases both -1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7DeltaR77-Y84 also stimulates +1 programmed frameshifting during prfB'-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7DeltaR77-Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K (1/2) for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism.

Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli.

The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535-1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540-1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535-1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535-1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.

The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation.

Herein, we describe an extracellular function of the vertebrate high-mobility group box 1 protein (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 functions as a DNA architectural protein, similar to the ubiquitous DNABII family of bacterial proteins; despite that, these proteins share no amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, whereas the DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. We showed that when both proteins converged on extracellular DNA within bacterial biofilms, HMGB1, unlike the DNABII proteins, disrupted biofilms both in vitro (including the high-priority ESKAPEE pathogens) and in vivo in 2 distinct animal models, albeit with induction of a strong inflammatory response that we attenuated by a single engineered amino acid change. We propose a model where extracellular HMGB1 balances the degree of induced inflammation and biofilm containment without excessive release of biofilm-resident bacteria.

The DNABII family of proteins is comprised of the only nucleoid associated proteins required for nontypeable Haemophilus influenzae biofilm structure.

Biofilms play a central role in the pathobiology of otitis media (OM), bronchitis, sinusitis, conjunctivitis, and pneumonia caused by nontypeable Haemophilus influenzae (NTHI). Our previous studies show that extracellular DNA (eDNA) and DNABII proteins are essential components of biofilms formed by NTHI. The DNABII protein family includes integration host factor (IHF) and the histone-like protein HU and plays a central role in NTHI biofilm structural integrity. We demonstrated that immunological targeting of these proteins during NTHI-induced experimental OM in a chinchilla model caused rapid clearance of biofilms from the middle ear. Given the essential role of DNABII proteins in maintaining the structure of an NTHI biofilm, we investigated whether any of the other nucleoid associated proteins (NAPs) expressed by NTHI might play a similar role, thereby serving as additional target(s) for intervention. We demonstrated that although several NAPs including H-NS, CbpA, HfQ and Dps are present within the biofilm extracellular matrix, only the DNABII family of proteins is critical for the structural integrity of the biofilms formed by NTHI. We have also demonstrated that IHF and HU are located at distinct regions within the extracellular matrix of NTHI biofilms formed in vitro, indicative of independent functions of these two proteins.