Biology subjects

Hobbs, J. K.

Publications and source records attributed to Hobbs, J. K..

11 recordsLinked to original sources

bioRxiv2026

Protein architectures of the bacterial spore envelope - common principles of assembly

Bacterial spores are among the most durable of cell forms, protected by robust envelopes of layered protein assemblies. Remarkably, these envelopes often share similar architectures across distantly related bacteria despite extensive divergence in their molecular components. How evolution has converged on such similar highly ordered and resilient cellular structures is relatively unexplored. Here, we combine targeted mutagenesis, cryo-electron microscopy, atomic force microscopy and structure prediction to determine the organisation and assembly of the outer spore envelope of Clostridium sporogenes, a genetically tractable surrogate for Group I Clostridium botulinum. We reveal a hierarchy of protein structures, including a semi-permeable two-dimensional crystalline exosporium; CsxA forms the exosporium scaffold with an outer "hairy nap" partially composed of BclA. We observe a previously uncharacterised multilayered three-dimensional crystalline parasporal assembly within the interspace between exosporium and coat (CsxC). These distinct structures are built from related cysteine-rich SPOCS (SpoVID-CotE-SipL)-domain proteins that self-assemble into highly ordered lattices. Such crystalline organisation, combined with high symmetry, can provide a template for local enhancement of cysteine concentration, favouring cooperative disulphide cross-linking and the formation of exceptionally stable supramolecular structures. Unexpectedly, the three-dimensional CsxC structure grows through screw dislocations, a mechanism familiar from inorganic and synthetic crystal growth but rarely demonstrated in native biological assemblies, with the exception of some biomineralisation processes. Our observations now suggest that this classical crystal-growth mechanism can be exploited in both mineralised and proteinaceous biological materials. Related SPOCS-domain proteins are implicated in spore-envelope assembly across diverse Clostridia, where they have diversified to act as both structural components and morphogenetic organisers. Remarkably, distantly related Bacilli construct similarly highly symmetric crystalline, cysteine-rich and disulphide-stabilised spore layers using proteins with very different protein folds. Thus, crystallisation and cooperative disulphide formation appear to represent a convergent physicochemical strategy for building diverse self-assembling proteins into exceptionally robust cellular assemblies. Together these findings provide the first molecular framework for understanding the organisation and assembly of the Clostridium spore envelope and reveal previously unrecognised principles governing the evolution of protective proteinaceous structures across the Bacillota (Firmicutes).

microbiology↗

bioRxiv2026

Morphological transformation in Helicobacter pylori is a dynamic process leading to two types of coccoid

The helical shape of Helicobacter pylori is crucial for successful colonization of the human stomach. However, this pathogen can shapeshift into another form termed the "coccoid form" with a spherical shape, through a mechanism that remains elusive. Here, by a combination of fluorescence microscopy using fluorescent D-aminoacids, cryoelectron and atom force microscopy, we explored the dynamics of coccoid formation in H. pylori through interrogation of the peptidoglycan layer. Contrary to the widely held hypothesis, we showed that helical-rod H. pylori transformed into a coccoid without transiting through a U-form. We show that U-forms, characterized by a U-shaped peptidoglycan with enlarged periplasmic space, altered genetic material, and red autofluorescence, are the output of a parallel pathway, which, unlike the coccoid pathway, is independent of the HdpA/Csd3 peptidoglycan endopeptidase. Coccoid formation occurred along a rigid timeline, by bulging of the cytoplasmic membrane through a peptidoglycan crack, resulting in a spheroplast-like structure with the peptidoglycan stacked into a thick layer near the original cell poles. Resistance of that structure against lysis likely involves a switch in metabolic profile reminiscent of bacteria in dormancy, with a notable accumulation of lysophospholipids, demonstrated in this work. Altogether, the ultrastructure and properties of H. pylori coccoids evidenced here are compatible with a role of this form in relapse after antibiotic treatment. SIGNIFICANCE STATEMENTUpon prolonged growth, stomach pathogen Helicobacter pylori undergoes a morphological change from a helical to a spherical form called coccoid, which may be involved in bacterial persistence and immune evasion. The pathway leading to this form, as well as its precise architecture, were unclear. In this work, we show by different microscopy techniques that the transition to coccoid does not involve a U-shaped intermediate as proposed before, but is triggered by progressive thinning, due to the activity of endopeptidase HdpA/Csd3, of the peptidoglycan meshwork that normally protects the cell, eventually leading to rupture. Because of the hole thus created, peptidoglycan can no longer contain the osmotic pressure in the cytoplasm, which leaks out within a membrane bulge, to eventually give rise to a kind of sphaeroplast, expected to be insensitive to cell wall-targeted antibiotics.

microbiology↗

bioRxiv2025

Clinical stringent response activation promotes conjugal transfer of staphylococcal resistance plasmids

Conjugative transfer of plasmids represents a major route through which antibiotic resistance genes are spread. In the case of the prevalent and deadly pathogen Staphylococcus aureus, >90% of clinical isolates carry at least one plasmid. While plasmid-encoded mechanisms (e.g. plasmid copy number) can influence conjugation frequency, host factors and environmental stimuli can also affect transmission. In particular, stress responses like the stringent response have been associated with increased movement of mobile genetic elements. We have previously shown that clinical mutations in the stringent response controller, Rel, lead to elevated levels of the alarmones (p)ppGpp and antibiotic tolerance in S. aureus. Here, we report that stringent response activation in these strains promotes the conjugal transfer of diverse staphylococcal resistance plasmids. We observed that clinical Rel mutations promote donation, but not receipt, of plasmids from the three families of staphylococcal plasmid and a mobilisable plasmid. This increased conjugation frequency could also be induced by chemical induction of the stringent response by mupirocin. Intriguingly, detailed experimental analysis revealed that the effect of elevated (p)ppGpp on plasmid donation was not due to CodY derepression, SOS response induction, increased plasmid copy number or increased expression of conjugation machinery genes. Further, transcriptomic analysis failed to identify any other putative plasmid- or host-derived mechanisms to explain this observation. Further investigations are required to explore the mechanistic link between the stringent response and conjugation, given the pervasive transcriptional and post-translational effects of (p)ppGpp. Overall, the association between Rel mutation and increased plasmid donation is alarming, especially as Rel mutations are being increasingly identified among clinical isolates.

microbiology↗

bioRxiv2025

The complete {lambda}-carrageenan depolymerization cascade from a marine Pseudoalteromonad revealed by structural analysis of the enzymes.

Carrageenans are a complex group of polysaccharides derived from the cell walls of red macroalgae. They are an abundant, yet recalcitrant nutrient source for most marine heterotrophic bacteria. Some member species of the Pseudoalteromonas genus are effective at metabolizing carrageenan. However, the enzymatic pathway for {lambda}-carrageenan, one of the most sulfated naturally occurring polysaccharides, remains unknown. Using detailed structural analysis by X-ray crystallography we reveal the sophisticated and cyclic enzymatic cascade deployed by Pseudoalteromonas distincta (strain U2A) to utilize {lambda}-carrageenan. The cascade incorporates ten glycoside hydrolases and five sulfatases that are specific for {lambda}-carrageenan and cooperate to completely deconstruct this polysaccharide, thus yielding galactose monosaccharides for subsequent energy production. The detailed molecular understanding of {lambda}-carrageenan depolymerisation provided includes structural evidence for a lesser described sulfatase catalytic mechanism and elucidation of a distinct catabolic cascade that is unique from previously described carrageenan metabolic pathways. This insight also holds potential for the application of enzymatic logic in the generation of high value products from abundant natural biopolymers, such as carrageenans.

biochemistry↗

bioRxiv2024

Staphylococcus aureus COL: An Atypical Model Strain of MRSA that Exhibits Slow Growth and Antibiotic Tolerance

Methicillin-resistant Staphylococcus aureus (MRSA) has been a pathogen of global concern since its emergence in the 1960s. As one of the first MRSA strains isolated, COL has become a common model strain of S. aureus. Here we report that COL is, in fact, an atypical strain of MRSA that exhibits slow growth and multidrug tolerance. Genomic analysis identified three mutated genes in COL (rpoB, gltX and prs) with links to tolerance. Allele swapping experiments between COL and the closely related, non-tolerant Newman strain uncovered a complex interplay between these genes. However, Prs (phosphoribosyl pyrophosphate [PRPP] synthetase) accounted for most of the growth and tolerance phenotype of COL. Biochemical and transcriptomic analysis revealed that COL does not exhibit slow growth as a result of partial stringent response activation, as previously proposed. Instead, the COL Prs mutation greatly reduces the PRPP synthetase activity of the enzyme and leads to downregulation of pyrimidine, histidine and tryptophan synthesis, three pathways that rely on PRPP. Overall, our findings indicate that COL is an atypical, antibiotic-tolerant strain of MRSA whose isolation predates the previous first report of tolerance among clinical isolates. Characterisation of clinical Prs mutations and their relationship with tolerance requires further investigation.

microbiology↗

bioRxiv2024

Flap endonuclease Substrate Interactions studied using Dynamic Single-Molecule Atomic Force Microscopy

Flap endonucleases (FENs) recognise and cleave DNA substrates containing a 5-single-strand (ss) of nucleic acid branching off a double-stranded (ds) DNA to yield a nicked duplex during DNA replication. Dynamic Atomic Force Microscopy of an inactive FEN mutant complexed with branched DNA revealed mobilisation of immobilised DNA, indicating that protein interaction affected substrate conformation and disrupted the forces that anchored it to the poly-L-ornithine -treated mica surface. Enzymatically-active FEN was seen intermittently binding DNA, altering its conformation and cleaving the ssDNA branch. We developed a method using motion tracking for quantifying the movement of DNA sections, by visually segmenting DNA and tracking each segment to recognise the DNA sections most affected by the protein. It was found that whilst bound, FEN caused localised DNA bending, and changes in DNA shape were witnessed in the short time span of the proteins appearance close to the nucleic acid, followed by protein adsorption on the mica surface. The results provide the first dynamic observations of FEN-DNA interaction. FEN initially binds to the dsDNA, slides to find the ds/ssDNA junction, and the 5 ssDNA likely threads through a hole in the enzyme which leads to enzymatic hydrolysis of the branched substrate.

biochemistry↗

bioRxiv2023

The roles of GpsB and DivIVA in Staphylococcus aureus growth and division

The spheroid bacterium S. aureus is often used as a model of morphogenesis due to its apparent simple cell cycle. S. aureus has many cell division proteins that are conserved across bacteria alluding to common functions. However, despite intensive study we still do not know the roles of many of these components. Here we have examined the functions of the paralogues DivIVA and GpsB in the S. aureus cell cycle. Cells lacking gpsB display a more spherical phenotype than wild type, associated with a decrease in peripheral cell wall peptidoglycan synthesis. This correlates with an increased localisation of penicillin binding proteins at the developing septum, notably PBPs 2 and 3. Our results highlight the role of GpsB as an apparent regulator of cell morphogenesis in S. aureus.

microbiology↗

bioRxiv2021

PBP1 of Staphylococcus aureus has multiple essential functions in cell division

Bacterial cell division is a complex process requiring the coordination of multiple components, to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multi- stage PG architecture that develops during septation. Penicillin binding proteins (PBPs) are essential for the final steps of PG biosynthesis - their transpeptidase activity links together the peptide sidechain of nascent glycan strands together. PBP1 is required for cell division in S. aureus and here we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thus coordinate the cell division process. The transpeptidase activity of PBP1 is also essential but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific {beta}-lactam, meropenem. Together these results lead to a model for septal PG synthesis where PBP1 enzyme activity is responsible for the characteristic architecture of the septum and PBP1 protein molecules coordinate cell division allowing septal plate formation.

microbiology↗

bioRxiv2021

Atomic force microscopy reveals the mechanical properties of breast cancer bone metastases

Mechanically dependent processes are essential in cancer metastases. However, reliable mechanical characterisation of metastatic cancer remains challenging whilst maintaining the tissue complexity and an intact sample. Using atomic force microscopy, we quantified the micro-mechanical properties of relatively intact metastatic breast tumours and their surrounding bone microenvironment isolated from mice, and compared with other breast cancer models both ex vivo and in vitro. A unique mechanical distribution of extremely low elastic modulus and viscosity was identified on metastatic tumours, which were significantly more compliant than both 2D in vitro cultured cancer cells and subcutaneous tumour explants. The presence of mechanically distinct metastatic tumour did not result in alterations of the mechanical properties of the surrounding microenvironment at meso-scale distances (> 200 {micro}m). These findings demonstrate the utility of atomic force microscopy in studies of complex tissues and provide new insights into the mechanical properties of cancer metastases in bone.

cancer biology↗

bioRxiv2020

Mechanical Heterogeneity in the Bone Microenvironment as Characterised by Atomic Force Microscopy

Bones are structurally heterogeneous organs with diverse functions that undergo mechanical stimuli across multiple length scales. Mechanical characterisation of the bone microenvironment is important for understanding how bones function in health and disease. Here we describe the mechanical architecture of cortical bone, the growth plate, metaphysis and marrow in fresh murine bones, probed using atomic force microscopy in physiological buffer. Both elastic and viscoelastic properties are found to be highly heterogeneous with moduli ranging over 3 to 5 orders of magnitude, both within and across regions. All regions include extremely soft areas, with moduli of a few Pascal and viscosities as low as tens Pa*s. Aging impacts the viscoelasticity of the bone marrow strongly but has limited effect on the other regions studied. Our approach provides the opportunity to explore the mechanical properties of complex tissues at the length scale relevant to cellular processes and how these impact on aging and disease. SIGNIFICANCEThe mechanical properties of biological materials at cellular scale are involved in guiding cell fate. However, there is a critical gap in our knowledge of such properties in complex tissues. The physiochemical environment surrounding the cells in in-vitro studies differs significantly from that found in vivo. Existing mechanical characterisation of real tissues are largely limited to properties at larger scales, structurally simple (e.g. epithelial monolayers) or non-intact (e.g. through fixation) tissues. In this paper, we address this critical gap and present the micro-mechanical properties of the relatively intact bone microenvironment. The measured Youngs moduli and viscosity provide a sound guidance in bioengineering designs. The striking heterogeneity at supracellular scale reveals the potential contribution of the mechanical properties in guiding cell behaviour.

biophysics↗

bioRxiv2020

Architecture and self-assembly of the Clostridium sporogenes/botulinum spore surface illustrate a general protective strategy across spore formers

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives have similar spore architectures incorporating protective proteinaceous envelopes. We reveal in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other Clostridial relatives, forms a hexagonally symmetric molecular filter. A cysteine-rich protein, CsxA, when expressed in E. coli, self-assembles into a highly thermally stable structure identical to native exosporium. Like exosporium, CsxA arrays require harsh reducing conditions for disassembly. We conclude that in vivo, CsxA self-organises into a highly resilient, disulphide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar in Bacillus spores, despite lack of protein homology. In both cases, intracellular disulphide formation is favoured by the high lattice symmetry. We propose that cysteine-rich proteins identified in distantly related spore formers may adopt a similar strategy for intracellular assembly of robust protective structures.

microbiology↗