Graham C. Walker

A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance

Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

The Y-Family of DNA Polymerases

We would like to thank Tomoo Ogi for generating the unrooted phylogenetic tree shown in Figure 1Figure 1 and Junetsu Ito for his comments on our proposal.

How rhizobial symbionts invade plants: the Sinorhizobium – Medicago model

Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which the bacteria can enter the plant root. Once the bacteria have been endocytosed within a host-membrane-bound compartment by root cells, the bacteria differentiate into a new form that can convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen fixation are dependent on the microaerobic environment and other support factors provided by the plant. In return, the plant receives nitrogen from the bacteria, which allows it to grow in the absence of an external nitrogen source. Here, we review recent discoveries about the mutual recognition process that allows the model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate inside its host plant alfalfa (Medicago sativa) and the model host plant barrel medic (Medicago truncatula).

Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance

SUMMARY DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol ζ, Pol κ, Pol η, and Pol ι.

Molecular Determinants of a Symbiotic Chronic Infection

Rhizobial bacteria colonize legume roots for the purpose of biological nitrogen fixation. A complex series of events, coordinated by host and bacterial signal molecules, underlie the development of this symbiotic interaction. Rhizobia elicit de novo formation of a novel root organ within which they establish a chronic intracellular infection. Legumes permit rhizobia to invade these root tissues while exerting control over the infection process. Once rhizobia gain intracellular access to their host, legumes also strongly influence the process of bacterial differentiation that is required for nitrogen fixation. Even so, symbiotic rhizobia play an active role in promoting their goal of host invasion and chronic persistence by producing a variety of signal molecules that elicit changes in host gene expression. In particular, rhizobia appear to advocate for their access to the host by producing a variety of signal molecules capable of suppressing a general pathogen defense response.

Antibiotics induce redox-related physiological alterations as part of their lethality

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.

Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

The exo genes of Rhizobium meliloti are needed for the synthesis of an acidic exopolysaccharide, succinoglycan. We have assigned biosynthetic roles to the products of the exo genes by characterizing succinoglycan biosynthetic intermediates from exo mutant strains. We propose a model of succinoglycan biosynthesis in which the products of the exoY and exoF genes function in the addition of the first sugar, galactose, to the lipid carrier; the products of the exoA, exoL, exoM, exoO, exoU, and exoW genes function in subsequent sugar additions; and the product of the exoV gene functions in the addition of pyruvate. The products of the exoP, exoQ, and exoT genes are required for polymerization of the octasaccharide subunits or transport of the completed polymer.

A novel exopolysaccharide can function in place of the Calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti

We have found that R. meliloti strain Rm1021, which is known to synthesize a Calcofluor-binding exopolysaccharide (EPS I), also has a cryptic capacity to synthesize a second exopolysaccharide (EPS II). Structural analysis of EPS II has shown that it differs in many respects from EPS I. Genetic analysis indicates that EPS II synthesis requires the products of at least seven loci on the second symbiotic megaplasmid of R. meliloti, and is induced by a mutation, expR101, which causes increased transcription of these genes. Synthesis of EPS II suppresses the symbiotic defects of EPS I-deficient strains on Medicago sativa (alfalfa), but not on four other plants that are normally hosts for Rm1021. These observations suggest that structural features of bacterial exopolysaccharides are involved in the determination of host range. The implications of these results for models of exopolysaccharide function, such as serving as signals to the plant or shielding the bacteria from plant defense responses, are discussed.

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase η (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD′2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase κ, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10–15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the ‘steric gate’ residue of DinB (Phe13 → Val) and that of its archaeal homologue Dbh (Phe12 → Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.

Alfalfa Root Nodule Invasion Efficiency Is Dependent on Sinorhizobium meliloti Polysaccharides

The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.