Martin Buck

Biological nitrogen fixation, arsenic biotransformation and antibiotic resistance are conventionally studied separately, yet the environmental biofilm—bacteria′s dominant organisational state in soils, sediments and rhizospheres—supplies a shared physicochemical and genetic context in which the three can intersect. The extracellular polymeric substance (EPS) matrix imposes steep oxygen, redox and metabolite gradients across micrometre scales and demonstrably shelters oxygen‐sensitive nitrogenase, retains arsenic‐transforming enzymes (ArsC, ArsM and AioA) and elevates horizontal gene transfer (HGT) frequencies orders of magnitude above planktonic conditions. Individual circuits are genetically established in single organisms—RpoN coactivates pslA and nifA and the non‐coding RNA NfiS stabilises nifK and katB transcripts, in Pseudomonas stutzeri A1501—but their assembly into one programme linking surface commitment, diazotrophic activation and oxidative stress defence, with additional c‐di‐GMP and ArsR stimulon input under arsenic stress, is proposed, not demonstrated. Arsenic‐driven coselection of antibiotic resistance genes (ARGs) is the best‐supported limb, operating through cross‐resistance (RND efflux), co‐resistance (ars–ARG colocation on Tn21, Class 1 integrons, IncP/IncQ plasmids) and coregulation (ArsR stimulon, SOS‐derepressed intI1), whereas the proposal that diazotrophy‐derived ammonium supports S‐adenosyl‐l‐methionine supply for ArsM‐mediated methylation rests on gene coenrichment and rate correlation rather than direct flux measurement. This review integrates these processes into a single biofilm framework, classifies each proposed linkage by the evidence behind it, identifies candidate trifunctional mobile elements carrying ars, nif and ARG cassettes in hyperaccumulator rhizospheres and paddy soils and specifies the minimum spatially resolved single‐cell dataset (bacterial‐MERFISH, par‐seqFISH and SIP‐NanoSIMS) needed to test the proposed couplings.

RNA repair is critical for cellular function. The Rtc system maintains RNA integrity within the translational machinery of bacteria. In E. coli , Rtc expression enables cells to rescue growth and survive treatment by conferring transient resistance to ribosome-targeting antibiotics, yet the mechanisms underpinning this resistance remain obscure. Here, we present a computational model of Rtc-regulated repair of translational RNAs. Integrating model predictions with experimental validations, we uncover notable cell-to-cell heterogeneity in rtc expression that impacts on translational capacity, indicating that rtc may induce a form of heteroresistance. We moreover identify Rtc targets that may reduce the translational capacity of cells and so potentiate antibiotic effects. Our findings elucidate a complex response underpinning resistance conferred by Rtc, offering alternate routes for addressing resistance in E. coli and other relevant pathogens.

Biosensors exploit the capabilities of biological systems to acquire a huge variety of chemical or physical information and convert molecular signals into actionable data. Here we took a bottom-up synthetic biology approach to combine the versatility and programmability of whole-cell bacterial biosensors with the sensitivity of electrochemical sensing devices. We built genetic modules to produce different phenazines and wired these to various sensing and information processing modules. A whole-cell bioelectronic sensor with a T7 RNAP-based signal amplifier was first constructed that detected mercury contaminants below the level of WHO safe limit for drinking water. We demonstrated the modularity and programmability of the sensor design by incorporating Boolean logic computation into a dual-input sensor. We subsequently engineered a sensor strain that can produce two phenazine types, giving a two-channel electrochemical output signal based on the detection of differentiated midpoint potentials. Our modular bioelectronic sensor therefore can be readily adapted for different applications and forms the basis for development of low-cost, field-deployable sensing devices.

Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use σ factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized σ factor, σ ⁵⁴ . σ ⁵⁴ recruits RNAP to promoters of genes associated with stress responses and forms a stable closed complex that does not spontaneously isomerize to the open state where promoter DNA is melted out and competent for transcription. The σ ⁵⁴ -mediated open complex formation requires specific AAA+ proteins ( A TPases A ssociated with diverse cellular A ctivities) known as bacterial enhancer-binding proteins (bEBPs). We have now obtained structures of new intermediate states of bEBP-bound complexes during transcription initiation, which elucidate the mechanism of DNA melting driven by ATPase activity of bEBPs and suggest a mechanistic model that couples the Adenosine triphosphate (ATP) hydrolysis cycle within the bEBP hexamer with σ ⁵⁴ unfolding. Our data reveal that bEBP forms a nonplanar hexamer with the hydrolysis-ready subunit located at the furthest/highest point of the spiral hexamer relative to the RNAP. ATP hydrolysis induces conformational changes in bEBP that drives a vectoral transiting of the regulatory N terminus of σ ⁵⁴ into the bEBP hexamer central pore causing the partial unfolding of σ ⁵⁴ , while forming specific bEBP contacts with promoter DNA. Furthermore, our data suggest a mechanism of the bEBP AAA+ protein that is distinct from the hand-over-hand mechanism proposed for many other AAA+ proteins, highlighting the versatile mechanisms utilized by the large protein family.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ ⁷⁰ class, represented by the σ ⁷⁰ that regulates housekeeping genes. σ ⁵⁴ forms a class on its own and regulates stress response genes. Extensive studies on σ ⁷⁰ have revealed the molecular mechanisms of the σ ⁷⁰ dependent process while how σ ⁵⁴ transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ ⁵⁴ initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ ⁵⁴ and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ ⁵⁴ and upstream DNA, enabling the transition to elongation.

Minimizing arsenic (74.92As33) loading into rice plants, we suggest adopting alternating wetting and drying (AWD) irrigation as a sustainable water management strategy allowing greater silicon (28.08Si14) availability. This two-year field-based project is the first report on AWD's impact on As-Si distribution in fluvio-alluvial soils of the entire Ganga valley (24 study sites divided into six Gangetic divisions), seasonal variance (pre-monsoon and monsoon), rice plant anatomy and productivity, soil microbial diversity, microbial gene ontology profiling, and cellular metabolic pathways. Under AWD to flooded and pre-monsoon to monsoon cultivations, respectively, As bioavailability was reduced by 8.71–9.25% and 25.68–26.13%. In the pre-monsoon and monsoon seasons, the rice grain yield under AWD improved by 8.41% and 9.96%, respectively proving the economic profitability. Compared to waterlogging, AWD proves to be the optimal soil condition for supporting soil microbial communities in rice fields, allowing diverse metabolic activities, As-resistance, and active expression of As-responsive genes and gene products. Finally, it is determined that by using AWD, the use of groundwater can be reduced, lowering the cost of pumping and field management and generating an economic profit for farmers. These combined assessments prove the acceptability of AWD for the establishment of multiple sustainable development goals (SDGs).

Nitrogen is an essential micronutrient for both plant and animal life and naturally exists in both reactive and inert chemical forms. Modern agriculture is heavily reliant on nitrogen that has been “fixed” into a reactive form via the energetically expensive Haber-Bosch process, with significant environmental consequences.

Farming of barley and chickpea is nitrogen (N) fertilizer dependent. Using strategies that increase the nitrogen use efficiency (NUE) and its components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE) would reduce the N fertilizer application in the soil and its adverse environmental effects. We evaluated the effects of three different strains of diazotroph Klebsiella (K.p. SSN1, K.q. SGM81, and K.o. M5a1) to understand the role of biological nitrogen fixation (BNF) and bacterial indole-3-acetic acid (IAA) on NUE of the plants. A field study revealed that K.p. SSN1 results in profound increment of root surface area by eightfold and threefold compared to uninoculated (control) in barley and chickpea, respectively. We measured significant increase in the plant tissue nitrogen, chlorophyll content, protein content, nitrate reductase activity, and nitrate concentration in the inoculated plants (p ≤ 0.05). Treated barley and chickpea exhibited higher NUE and the components compared to the control plants (K.p. SSN1 ≥ K.q. SGM81> K.o. M5a1). Specifically, K.q. SGM81 treatment in barley increased NUpE by 72%, while in chickpea, K.p. SSN1 increased it by 187%. The substantial improvement in the NUpE and NUE by the auxin producers K.p. SSN1 and K.q. SGM81 compared with non-auxin producer K.o. M5a1 was accompanied by an augmented root architecture suggesting larger contribution of IAA over marginal contribution of BNF in nitrogen acquisition from the soil.