Biology subjects

Webb, J. S.

Publications and source records attributed to Webb, J. S..

7 recordsLinked to original sources

bioRxiv2024

Simulating Offshore Oilfield Conditions: Insights into Microbiologically Influenced Corrosion from a Dual Anaerobic Biofilm Reactor

Oilfield systems are a multifaceted ecological niche which consistently experience microbiologically influenced corrosion. However, simulating environmental conditions of an offshore system within the laboratory is notoriously difficult. A novel dual anaerobic biofilm reactor protocol allowed a complex mixed-species marine biofilm to be studied. Interestingly, electroactive corrosive bacteria and fermentative electroactive bacteria growth was supported within the biofilm microenvironment. Critically, the biotic condition exhibited pits with a greater average area which is characteristic of microbiologically influenced corrosion. This research seeks to bridge the gap between experimental and real-world scenarios, ultimately enhancing the reliability of biofilm management strategies in the industry. ImportanceIt is becoming more widely understood that any investigation of microbiologically influenced corrosion requires a multidisciplinary focus on multiple lines of evidence. While there are numerous standards available to guide specific types of testing, there are none that focus on integrating biofilm testing. By developing a novel dual anaerobic reactor model to study biofilms, insights into the different abiotic and biotic corrosion mechanisms under relevant environmental conditions can be gained. Using multiple lines of evidence to gain a holistic understanding, more sustainable prevention and mitigation strategies can be designed. To our knowledge, this is the first time all these metrics have been combined in one unified approach. The overall aim for this paper is to explore recent advances in biofilm testing and corrosion research, to provide recommendations for future standards being drafted. However, it is important to note that this article itself is not intending to serve as a standard.

microbiology↗

bioRxiv2023

A redox switch allows binding of ferrous and ferric ions in the cyanobacterial iron binding protein FutA from Prochlorococcus

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance StatementOceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

biophysics↗

bioRxiv2022

Multi-excitation Raman Spectroscopy Complements Whole Genome Sequencing for Rapid Detection of Bacterial Infection and Resistance in WHO Priority Pathogens

Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.

microbiology↗

bioRxiv2020

An integrated model system to gain mechanistic insights into biofilm formation and antimicrobial resistance development in Pseudomonas aeruginosa MPAO1

Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and missed genes by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq datasets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth. Experiments conducted across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated both known and novel genes involved in biofilm growth and antibiotic resistance identified in screens of the mutant collection. Differential protein expression data from planktonic cells versus biofilm confirmed upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type six secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance and resistance evolution.

systems biology↗

bioRxiv2020

Antimicrobial activity of the quinoline derivative HT61, effective against non-dividing cells, in Staphylococcus aureus biofilms

Staphylococcus aureus is an opportunistic pathogen responsible for a wide range of chronic infections. Disease chronicity is often associated with biofilm formation, a phenotype that confers enhanced tolerance towards antimicrobials, a trait which can be attributed to a dormant, non-dividing subpopulation within the biofilm. Development of antibiofilm agents that target these populations could therefore improve treatment success. HT61 is a quinoline derivative that has demonstrated efficacy towards non-dividing planktonic Staphylococcus spp. and therefore, in principal, could be effective against staphylococcal biofilms. In this study HT61 was tested on mature S. aureus biofilms, assessing both antimicrobial efficacy and characterising the cellular response to treatment. HT61 was found to be more effective than vancomycin in killing S. aureus biofilms (minimum bactericidal concentrations: HT61; 32 mg/L, vancomycin; 64 mg/L), and in reducing biofilm biomass. Scanning electron microscopy of HT61-treated biofilms also revealed disrupted cellular structure and biofilm architecture. HT61 treatment resulted in increased expression of proteins associated with the cell wall stress stimulon and dcw cluster, implying global changes in peptidoglycan and cell wall biosynthesis. Altered expression of metabolic and translational proteins following treatment also confirm a general adaptive response. These findings suggest that HT61 represents a new treatment for S. aureus biofilm-associated infections that are otherwise tolerant to conventional antibiotics targeting actively dividing cells.

microbiology↗

bioRxiv2019

Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa

Bacteria typically occur either as free-swimming planktonic cells or within a sessile, biofilm mode of growth. In Pseudomonas aeruginosa, the transition between these lifestyles is known to be modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP). We are interested in the control of distinct biofilm-relevant phenotypes in P. aeruginosa through the modulation of intracellular c-di-GMP. Here, we characterise motility and associated biofilm formation and dispersal in two pairs of related multi-domain proteins with putative c-di-GMP turnover domains, selected to contain additional PAS (Per-Arnt-Sim) homology domains known for their ability to process environmental stimuli. The enzymes PA0861 (RbdA) and PA2072 have distinct functions despite their similar domain structures. The {Delta}rbdA deletion mutant showed significantly increased biofilm formation while biofilm formation was impaired in {Delta}PA2072. Using a GFP transcriptional reporter fused to the cyclic di-GMP-responsive cdrA promoter, we show correlation between biofilm phenotype and c-di-GMP levels. Both proteins are shown to play a role in nitric oxide (NO) induced biofilm dispersal. We further studied pseudo-enzymes of similar architecture. PA5017 (DipA) is an inactive cyclase, and PA4959 (FimX) is described here as an inactive cyclase/phosphodiesterase. Loss of swimming and twitching motilities, respectively, is observed in deletion variants, which correlated with NO-induced biofilm dispersal phenotypes, as {Delta}dipA dispersed less well while {Delta}fimX dispersed better than wild type. The study highlights how Pseudomonas differentiates c-di-GMP output - in this case motility - using structurally very similar proteins and underlines a significant role for pseudo-enzymes in motility regulation and associated biofilm dispersal.\n\nImportanceBacterial biofilms exert pervasive economic and societal impact across a range of environmental, engineered and clinical contexts. The secondary messenger cyclic guanosine di-phosphate, c-di-GMP, is known to control the ability of many bacteria to form biofilms. The opportunistic human pathogen Pseudomonas aeruginosa PAO1 has 38 putative enzymes that can regulate c-di-GMP turnover, and these proteins modulate various cellular functions and influence bacterial lifestyle. The specific protein sensory domains and mechanisms of motility that lead to biofilm dispersal remain to be fully understood. Here we studied multi-domain proteins with the PAS (Per-Arnt-Sim) homology domains, these being classic sensors to environmental stimuli. Our study demonstrates the significant roles for the pseudo-enzymes PA4959 (FimX) and PA5017 (DipA) in regulation of biofilm phenotype and motility. Further, enzymes with highly homologous structures, such as PA0861 (RbdA) and PA2072, have almost orthogonal function in biofilm and motility control.

microbiology↗

bioRxiv2018

Increased rates of genomic mutation in a biofilm co-culture model of P. aeruginosa and S. aureus

Biofilms are major contributors to disease chronicity and are typically multi-species in nature. Pseudomonas aeruginosa and Staphylococcus aureus are leading causes of morbidity and mortality in a variety of chronic diseases but current in vitro dual-species biofilms models involving these pathogens are limited by short co-culture times (24 to 48 hours). Here, we describe the establishment of a stable (240 hour) co-culture biofilm model of P. aeruginosa and S. aureus that is reproducible and more representative of chronic disease.\n\nThe ability of two P. aeruginosa strains, (PAO1 and a cystic fibrosis isolate, PA21), to form co-culture biofilms with S. aureus was investigated. Co-culture was stable for longer periods using P. aeruginosa PA21 and S. aureus viability within the model improved in the presence of exogenous hemin. Biofilm co-culture was associated with increased tolerance of P. aeruginosa to tobramycin and increased susceptibility of S. aureus to tobramycin and a novel antimicrobial, HT61, previously shown to be more effective against non-dividing cultures of Staphylococcal spp. Biofilm growth was also associated with increased short-term mutation rates; 10-fold for P. aeruginosa and 500-fold for S. aureus.\n\nBy describing a reproducible 240 hour co-culture biofilm model of P. aeruginosa and S. aureus, we have shown that interspecies interactions between these organisms may influence short-term mutation rates and evolution, which could be of importance in understanding the adaptive processes that lead to the development of antimicrobial resistance.

microbiology↗