Journal of Bacteriology

Bacteria in natural environments frequently encounter nutrient limitation leading to growth arrest and must balance the potential benefits of continuing to respond to the environment by making new proteins against the costs of depleting limited resources. We previously showed that the RNA polymerase-binding regulator SutA enhances transcription of hundreds of genes during nutrient limitation in Pseudomonas aeruginosa, suggesting that it might be part of a regulatory network facilitating limited new protein synthesis. Here, we sought to expand our understanding of this network by identifying transcriptional regulators influencing sutA expression. Using northern blotting, western blotting, and reporter assays, we found that the sigma factors FliA and RpoS, and the DNA-binding regulator Lrp, impact expression from a proximal sutA promoter during the transition to stationary phase. This constellation of regulators and the dynamics of SutA expression lead us to propose that SutA is part of a regulatory network that facilitates scavenging. Scavenging includes motility toward possible nutrient sources and uptake mechanisms for these nutrients, activities which require an investment of resources but can yield important benefits during starvation. In vitro transcription experiments, proteomic analysis and reporter assays suggest that SutA directly supports new protein synthesis driven by RpoS and indirectly supports flagellar motility, perhaps by helping maintain protein biosynthetic capacity against the metabolic costs of motility. SutA expression is controlled by multiple regulatory inputs, including negative autoregulation, and the protein appears to be short-lived. These properties are consistent with a role in supporting short, controlled bursts of gene expression during nutrient limitation. Author StatementMany bacteria engage in cycles of colonising a nutrient-rich location, using the available nutrients, and then dispersing in search of a new location to colonise. While searching for new nutrients in a low-resource environment, bacteria will be starved and must coordinate resource-intensive processes such as new protein synthesis, motility, and nutrient uptake so that each crucial activity can be accomplished but none use too much of the limited pool of resources. We previously identified a regulator in Pseudomonas aeruginosa called SutA, which facilitates new protein synthesis under starvation conditions. Here, we have identified regulators of SutA expression. We find that the housekeeping sigma factor RpoD drives expression during growth, but at the entry to stationary phase, where SutA has obvious impacts on cellular physiology, the stress sigma factor RpoS, the flagellar sigma factor FliA, and the amino acid sensing transcription factor Lrp are important. Finally, we find that all cells in a nutrient-limited population express some SutA, but appear to do so in infrequent bursts, and that the protein is likely unstable. Together, these findings suggest that SutA contributes to the coordination of resource use while bacteria scavenge for new nutrients, facilitating limited amounts of new protein synthesis.

The horizontally acquired mgtC gene from Salmonella enterica confers this bacterium the abilities to survive episodes of magnesium (Mg2+) starvation, and to replicate in mammalian macrophages. The former property allows bacteria to persist in the environment through periods of Mg2+ depletion, whereas the latter allows S. enterica to overcome self-limiting intestinal colonization and cause an invasive systemic infection in susceptible mammalian hosts. Even though the biochemical function of MgtC is not completely understood, this protein is thought to function primarily by preventing the production of toxic levels of Mg2+-chelating adenosine triphosphate (ATP). In the current work, we investigated the physiological roles of mgtC homologs from an array of bacterial species, by probing the processes controlled by this gene during replication in low Mg2+ medium and in macrophages. We determined that MgtC homologs that do not participate in Pi homeostasis during Mg2+ starvation and do not promote intramacrophage replication in their resident species can partake in these processes when expressed in S. enterica. This indicates that the function of this protein is context dependent. Accordingly, we show that the physiological processes affected by S. enterica MgtC vary, depending on whether the bacteria replicate in low Mg2+ medium or inside macrophages. While these results suggest that MgtC is a regulator, they also demonstrate that horizontally acquired genes can assume different roles, depending on the genome and the biochemical context into which they are inserted. ImportanceThe mgtC gene encodes an inner membrane protein that has been horizontally acquired by multiple bacterial species, including several mammalian pathogens. In Salmonella enterica, MgtC promotes replication in mammalian macrophages and allows this bacterium to survive cytoplasmic magnesium (Mg2+) starvation. These phenotypes are thought to result from MgtCs inhibition of Pi metabolism and ATP production, which prevents the accumulation of toxic levels of Mg2+-chelating ATP and disrupts other physiological processes that are strictly dependent on Mg2+, such as ribosome assembly and translation. In the current study, we show that processes that are controlled by MgtC vary with the genetic and biochemical contexts in which this protein is expressed. While establishing a broader role for MgtC as a regulator, our findings illustrate how horizontally acquired regulatory genes can potentiate regulatory interactions, facilitating the evolution of new traits.

To establish infection and cause disease, the intracellular pathogen Shigella must successfully navigate a series of host defenses and distinct microenvironments within the human body. One way Shigella navigates these enviroments is by using the secondary messenger c-di-GMP, which regulates many different bacterial behaviours. C-di-GMP is synthesized by diguanylate cyclases (DGCs) and broken down by c-di-GMP specific phosphodiesterases (PDEs). In this study, we investigated how Shigellas c-di-GMP specific PDEs impact c-di-GMP turn-over and subsequently biofilm and virulence phenotypes. We knocked out each of Shigellas six c-di-GMP specific PDEs to determine how these PDEs impact biofilm, virulence and c-di-GMP levels within the bacterial cell. We found that these PDEs negatively regulate c-di-GMP levels while modulating Shigellas virulence and biofilm behaviour. We also noted that altering expression of these Shigella PDEs changes bacterial cell size. Transcriptome analysis revealed that a Shigella {Delta}pdeB strain showed reduced expression of many genes, including the virulence genes ipgD and ipgE, as well as genes associated with lipid metabolism. We confirmed that a Shigella {Delta}pdeB strain had altered levels of stearic acid, and expression of pdeB alters Shigella antibiotic susceptibility. This study highlights the complexities of c-di-GMP signaling in regulating numerous Shigella pathways.

In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). But in the opportunistic pathogen Clostridioides difficile, 70% of the crosslinks are 3-3 crosslinks formed by L,D-transpeptidases (LDTs), and LDTs are essential for viability. PBPs and LDTs use different acyl donors for crosslinking; PBPs require a pentapeptide, while LDTs require a tetrapeptide. Here, we determined the source of the tetrapeptides in C. difficile and investigated the consequences of reengineering PG crosslinking from predominantly 3-3 to exclusively 4-3. We found that two D-alanyl-D-alanine carboxypeptidases (DD-CPase), DacA and DacC, supply LDTs with tetrapeptides during vegetative growth. Deleting these enzymes was sufficient to bypass the normal requirement for LDTs. The resulting mutant ({Delta}dacAC {Delta}ldt) was remarkably healthy despite the absence of 3-3 crosslinks. Its only major phenotypic defect was a 3- to 4-log decrease in sporulation, which could, however, be overcome by deleting a third DD-CPase, dacB. These findings fill gaps in our understanding of the pathway for LD-transpeptidation in C. difficile and imply that LDTs are not essential components of the elongasome or divisome, both of which function well in the complete absence of LDTs, provided there is sufficient pentapeptide to sustain crosslinking by PBPs. Thus, LDTs are essential for viability because C. difficile has intrinsically high levels of DD-CPase activity. Finally, we propose a model for how PBPs and LDTs work together during PG synthesis. In this model, PBPs construct a sparsely crosslinked PG sacculus that is subsequently strengthened with crosslinks introduced by LDTs.

Bacteria frequently face challenges adapting to changing environmental conditions to survive and thrive, such as shifting resource utilization. Methylotrophs capable of growth on reduced single-carbon compounds are prevalent in the phyllosphere (aerial plant surfaces), where they face continual and predictable shifts in the availability of different plant-produced carbon sources. We examined the ability of the methylotroph Methylobacterium extorquens PA1 to adapt to repeated shifts between two different carbon and energy sources: the one-carbon compound methanol and the multi-carbon organic acid succinate, both present in the phyllosphere. Evolved lineages of wild-type cells all increased their capacity for rapid transition between the carbon sources through high frequencies of loss-of-function mutations affecting a previously uncharacterized gene, named cstR for carbon source transition regulator, which encodes an orphan single-domain response receiver. Characterization showed that mutant strains were more competitive bidirectionally in the succinate-methanol transition. Though evolved populations of the {Delta}efgA and {Delta}ttmR strains, which are defective in the succinate-to-methanol transition, experienced similar phenotypic improvements in carbon-source transitions, we did not observe cstR mutations rise to prominence as extensively or frequently in these lineages. Transcriptomic work revealed loss-of-function to cstR impacted expression of genes involved in motility/chemotaxis, energy metabolism, and stress response, among others, suggesting that it coordinates responses to metabolic cues that are prevalent in certain carbon source and growth phase transitions. Loss of cstR function did not compromise exogenous formaldehyde tolerance in the {Delta}efgA and {Delta}ttmR mutants, breaking a previously described tradeoff between these two phenotypes. However, this loss did lead to defects under exposure to certain stressors, including heat, desiccation, oxidative stress agents, and particularly pH stress. Altered levels of NAD+/NADH across conditions, improved growth under acidic pH, and diminished ATP and increased mortality under heightened pH together support a model where CstR is responsible for coordinating cell signaling to manage the balance between growth and maintaining stress resilience.

The Min system disassembles FtsZ-rings after septation in Bacillus subtilis and is localized to the nascent division plane and cell poles by the protein MinJ. The N-terminal region of MinJ contains transmembrane segments while the C-terminal region of MinJ contains a PDZ domain but its topology and functional domains are poorly understood. Here we empirically test MinJ topology based on a variety of transmembrane prediction models and find that the data is most consistent with Alphafold3, which predicts a 9-pass transmembrane protein with an external N-terminus and internal C-terminus. Deletion analysis indicates that all regions of the protein tested are required for function but deletion of the PDZ domain alone preserves polar localization and interaction with both MinD and DivIVA. Moreover, Alphafold predicts that transmembrane segments 6 and 7 comprise staves of an unusual transmembrane {beta}-sheet and deletion of the putative {beta}-sheet in the absence of MinD results in a minicell frequency that exceeds mutation of MinD alone. Bioinformatic analysis indicates that MinJ is highly conserved within Firmicutes and is co-conserved with MinD and DivIVA with which it interacts. Our data clarify the structure of MinJ and support models in which MinJ has functions in addition to restricting the activity of the Min system.

Biological ethanol production is important for the circular carbon economy and makes up 73% of the U.S. biological fuels market. Previously, we produced ethanol by reversing methanogenesis and capturing methane by cloning methyl-coenzyme M reductase (Mcr) from an unculturable population of anaerobic methanotrophic archaea; this process was predicated on the generation of the intermediate acetate and its conversion by the methanogenic host to ethanol. Moreover, methanogens are generally thought to be detrimental for converting acetate to ethanol and are usually intentionally inhibited. Here, we demonstrate that direct growth on acetate as the sole carbon and energy source by the methanogen Methanosarcina acetivorans C2A results in 40% of the metabolized acetate becoming ethanol and that there is 430% more ethanol produced, compared to growth on methane via Mcr. In addition, we found growth on methanol results primarily in methane generation and low levels of ethanol. Therefore, acetate may be readily converted by the methanogen M. acetivorans to ethanol at high yields.

Shigella sonnei is an enteropathogen that causes bacillary dysentery. During the first step of its virulence cycle, it must outcompete the resident microbiota to establish its own niche. Here we report that SigA, the sole SPATE (Serine Protease Autotransporter of Enterobacteriaceae) family member in S. sonnei, plays an indirect but central role in this process. A genome-wide analysis showed that the SPATE family includes SigA, Pic, SepA, and Sat. We demonstrated that SigA self-assembles into amyloid fibrils (F-SigA) independently of its protease activity. F-SigA remains associated with the S. sonnei surface in vitro and in vivo. Purified F-SigA fibrils have a diameter of 17.7 {+/-} 3.2 nm, and their amyloid organization was confirmed using specific markers and biochemical methods. F-SigA is secreted into the lumen in vivo and localizes to the surface of the colonic epithelium. We found that colicin E1 (ColE1) interacts with F-SigA amyloid fibrils, and that F-SigA-ColE1 complexes display antimicrobial activity that promotes S. sonnei competition with other bacteria. Because Pic, another Shigella SPATE, also forms amyloid fibrils, we anticipate that this virulence mechanism may be relevant across a wide range of Shigella strains and enterobacteria and may serve additional roles during the Shigella virulence cycle.

Rhodopseudomonas palustris TIE-1 (TIE-1) is a metabolically versatile environmental bacterium that flourishes across gradients of iron, oxygen, and light. This versatility necessitates extensive regulatory control, exemplified by the aerobic-anaerobic metabolic shift controlled by the hierarchy of CRP/FNR-family regulators AadR and FixK. Many anaerobic metabolic pathways demand expression of iron cofactor-intensive proteins, and TIE-1 in particular can generate energy through phototrophic iron oxidation via the PioABC system. However, TIE-1 lacks canonical iron-sensing regulators: IscR, ancestral Fe(II)-sensing Fur, and Fe(II)-sensing RirA of Rhizobiaceae, leaving it unclear how TIE-1 coordinates expression of these iron-requiring metabolisms with bioavailable iron levels. Here, we demonstrate that the AadR-FixK hierarchy plays a previously underappreciated role in iron regulation in TIE-1 by comparing growth and transcription in wild-type and regulatory mutants across wetland-inspired naturomimetic conditions. {Delta}aadR and {Delta}fixK showed defects in iron-dependent growth and Fe(II) oxidation, and the {Delta}aadR{Delta}fixK double mutant was synthetically lethal under anaerobiosis. The regulatory hierarchy of FixK and AadR influences expression of Fur-family regulators: the two irr paralogs were oppositely regulated in the presence of AadR, and absence of AadR perturbed iron-responsive expression of mur. Furthermore, the AadR regulon was significantly enriched for iron-related and iron-containing proteins. Despite initial predictions that AadR directly regulates pioABC, we found no conclusive evidence for direct AadR activity at the pioABC promoter, refining the search for pio regulators. Together, these findings establish AadR as a central integrator of oxygen and iron signals to coordinate iron-requiring anaerobic metabolism in TIE-1.

Type IV pili (T4P) mediate twitching motility and contribute to surface colonization, biofilm formation, and host interactions in Acinetobacter baumannii. However, the prevalence, dynamics, and diversity of twitching motility across A. baumannii populations remain poorly understood. Here, we compared twitching motility in a collection of 35 A. baumannii strains originating from clinical, environmental, and animal sources, using Pseudomonas aeruginosa PAO1 as a reference. Standardization of assay conditions revealed a strong influence of agar composition on twitching motility, with Eiken agar supporting the most robust surface translocation. Under these conditions, 14 of 35 A. baumannii isolates exhibited detectable twitching motility. Time-lapse microscopy revealed major differences between A. baumannii and P. aeruginosa. Whereas PAO1 initiated twitching within minutes after inoculation and formed characteristic multicellular rafts, motile A. baumannii strains displayed a prolonged non-motile phase before movement initiation and exhibited distinct patterns of collective organization. Two major expansion phenotypes were identified, termed Homogeneous Front (HF) and Raft-Like Front (RLF), together with Early-Onset Motility (EOM) and Delayed-Onset Motility (DOM) subgroups. Quantitative analyses further revealed substantial variation in speed, directional persistence, and migration dynamics among strains. Because a majority of isolates were non-motile, we investigated the contribution of the minor pilin FimT. Although deletion of fimT abolished twitching motility and specific substitutions modulated motility efficiency, sequence variation in FimT alone could not account for the observed phenotypic diversity. Collectively, these findings reveal extensive heterogeneity in T4P-mediated surface motility in A. baumannii and identify delayed twitching activation and distinct collective migration strategies as key features of surface colonization in this species.

Regulation of class A penicillin-binding proteins (aPBPs) in peptidoglycan biosynthesis is incompletely understood in Gram-positive bacteria. One example is activation of aPBP2a by GpsB and phosphorylated MacP in the ovoid-shaped pathogen, Streptococcus pneumoniae. We set out to examine whether phosphorylation of Thr residues other than Thr32 contributed to MacP activation of aPBP2a. We also wanted to determine whether GpsB and MacP activation of aPBP2a were related. Here we report that MacP was phosphorylated about equally at Thr32 and Thr56 in physiological and biochemical assays. However, based on transformation and growth assays, phosphorylation of MacP was not required for aPBP2a activation. A structure-function analysis confirmed that most of the MacP cytoplasmic domain, which was predicted by AlphaFold3 to be disordered, was not required for aPBP2a activation. These analyses further identified amino acids in the MacP transmembrane domain and the aPBP2a juxtamembrane region, as well as a variant of the GpsB-binding motif in the membrane-proximal cytoplasmic region of MacP, required for aPBP2a activation. Together, these results support a tripartite model in which GpsB acts as an adapter for activation of aPBP2a by MacP. Finally, additional interaction, Tn-seq, and growth assays suggested other modes of direct or indirect regulation of aPBP2a activity.

Bloom-forming cyanobacteria thrive in highly dynamic light environments, yet the mechanisms enabling rapid acclimation to fluctuating irradiance remain poorly understood. Here, we compared light acclimation in the bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 and the non-bloom-forming model cyanobacterium Synechocystis sp. PCC 6803 and investigated the role of the cyanobacterial toxin microcystin (MC) and its in vivo binding partner RubisCO in this process. Whereas Synechocystis grew faster under sustained high light, Microcystis performed better under low light and responded to transient high-light exposure with a remarkably rapid increase in photosynthetic activity and glycogen accumulation. These responses were markedly attenuated in an MC-deficient mutant. Although RubisCO from Microcystis exhibited pronounced light-dependent changes in activity, MC had only minor effects on RubisCO catalysis, arguing against a direct role in regulating enzyme function. Instead, extracellular MC elicited a transient transcriptional program characterized by induction of inorganic carbon acquisition systems, including the high-affinity bicarbonate transporter BCT1, consistent with activation of the carbon-concentrating mechanism (CCM) and enhanced carbon fixation in vivo. MC further stimulated the expression of photosynthesis-related genes, and altered carboxysome organization, and promoted extracarboxysomal localization of RubisCO. Together, our findings identify MC as a light-responsive signaling molecule that coordinates CCM activity, carbon acquisition, and photosynthetic acclimation, thereby enhancing adaptation of Microcystis to fluctuating irradiance and potentially contributing to its ecological success in cyanobacterial blooms.

The Type VI secretion system (T6SS) is a bacterial nanoweapon that injects toxic effectors into prokaryotic and eukaryotic cells. It is widely found among gram-negative bacteria and provides a significant fitness advantage in interbacterial competition. Pseudomonas putida KT2440 possesses three T6SS clusters (K1-, K2- and K3-T6SS) that combat phytopathogens. This makes this strain a potent biocontrol agent that protects plants from pathogens and can be further enhanced by a better understanding of its T6SS regulation. Although the core components of T6SS are conserved, the elements controlling its regulation differ among bacterial species. T6SS activity is regulated by various factors acting at different levels, from transcription to post-translational modification, to ensure precise control of its activity. Here, we demonstrate the critical importance that the three Rsm proteins, RsmIEA, have in controlling the K1-T6SS structural components and related orphan elements at the post-transcriptional level in Pseudomonas putida. We identified multiple Rsm-binding sites responsible for directly repressing the translation of T6SS proteins (Hcp1 and Hcp5) and their associated effectors (Tke2 and Tke7). Derepression of K1-T6SS mRNA in the rsmIEA mutant led to enhanced translation and expression of the K1-T6SS components and effectors, and critically increased the number of cells in the population with assembled T6SS. This results in a greater capacity to secrete toxins and kill prey cells via the T6SS-dependent mechanism. Finally, we demonstrate the K1-T6SS ability to kill environmental pathogens, including Salmonella enterica and Erwinia amylovora.

Small RNAs (sRNAs) rarely cause strong growth phenotypes upon overexpression, complicating efforts to link regulatory interactions to physiological outcomes. Here, we report that high levels of the Escherichia coli sRNA OmrA, but not its sibling OmrB, severely inhibit growth in glucose minimal medium. Genetic, biochemical, and physiological analyses indicate that OmrA-dependent toxicity results from reduced flux through the tricarboxylic acid (TCA) cycle. A UV-based suppressor screen identified mutations in the gene encoding Hfq, the RNA-chaperone that aids sRNA-mRNA interactions. Secondly, three independent mutations clustered in the ribosome-binding site of ppc, encoding phosphoenolpyruvate carboxylase, a key anaplerotic enzyme. OmrA directly inhibits Ppc translation via Hfq-dependent base-pairing in the ppc 5' UTR, including the mutated nucleotides obtained in the genetic screen. OmrA is significantly more effective than OmrB in ppc repression in vivo and in vitro, consistent with sequence divergence in their central regions. Supplementation with glutamate, glutamine, or downstream TCA cycle metabolites fully restores growth, linking reduced Ppc levels to metabolic limitation. These results identify ppc as a physiologically relevant OmrA target and suggest how RNA toxicity can uncover central metabolic nodes used by sRNAs to modulate bacterial physiology.

Endosymbiosis is widespread throughout the tree of life. Understanding how the transition of a bacterial endosymbiont from facultative to host-dependent obligate life occurs is an important question for defining the origin of endosymbiosis. A novel gram-positive bacillus, Brevibacillus sp. TJ4 was isolated from the nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1, which houses several endobacteria within its cells. TJ4 can survive independently of yeast but exhibits genomic and metabolic features characteristic of an evolving endosymbiont, slowly assuming a host-dependent, obligate lifestyle. The TJ4 genome contains several incomplete pathways for carbohydrate, amino acid, vitamin, and cofactor metabolism, which is reflected in its increased reliance on host-derived nutrients and auxotrophy compared with that of other Brevibacillus spp. Comparative genomics revealed widespread genome rearrangements, loss of synteny, and multiple cross-genus and inter-kingdom horizontal gene transfer (HGT) events in TJ4 compared to other Brevibacillus spp. These HGTs include the acquisition of genes from bacteriophages and co-resident endobacteria of JGTA-S1. One such horizontally acquired gene, Type II 3-dehydroquinate dehydratase (AroQ), appears to have originated from the Rhodotorula host itself. This acquisition functionally restores the shikimate pathway in strain TJ4, as evidenced by the phylogenetic placement of AroQ from TJ4 within the clade of fungal AroQ homologs. Potential exploitation of the host JGTA-S1 appears to be a probable mode of endosymbiosis of TJ4, an evolving endosymbiont that we named Brevibacillus rhodotorulae sp. nov.

First described pyrroloquinoline quinone-dependent (PQQ) eight-bladed {beta}-propeller proteins are calcium (Ca)-dependent, but many homologous bacterial enzymes are lanthanide (Ln)-dependent. Discovery of Ca-dependent six-bladed {beta}-propeller PQQ-dependent dehydrogenases motivated the search for Ln-dependent six-bladed {beta}-propeller PQQ-dependent enzymes in bacteria. Using in silico structural prediction of sequences from weathered rock, we identified [~]22,000 PQQ six-bladed {beta}-propeller proteins in bacteria from 77 phyla, of which 63% of sequences have the active site residues needed to bind Ln. PQQ and La binding was biochemically confirmed for enzymes from uncultivated Chloroflexi and Acidobacteria. In structural models, Ln-binding periplasmic proteins interact with TonB-dependent transporters that may enable Ln uptake. Most genomes also encode predicted Ln-and PQQ-dependent eight-bladed dehydrogenases that clade with diverse alcohol and sugar dehydrogenases. Thus, Ln-dependent six and eight-bladed PQQ-dependent {beta}-propeller proteins are implicated in diverse carbon substrate metabolisms in weathering rock and soil. We predict that many PQQ-dependent microbial enzymes are lanthanide dependent.

The architecture of Bacillus subtilis biofilms is influenced by the coordinated regulation of cellular specialization, matrix assembly, and metabolism. B. subtilis can form different types of biofilm in diverse physical and chemical environments. Understanding the molecular mechanisms that drive biofilm heterogeneity and adaptation to different environmental niches is crucial for developing more effective strategies to control their formation. In this study, we developed a tightly dual-regulated CRISPR interference (CRISPRi) system and employed multi-scale imaging to investigate the functions of individual genes in two distinct biofilm models: the floating pellicle and the intricate, three-dimensionally structured macrocolony, which develop at the liquid-air and solid-air interfaces, respectively. Our findings validated the CRISPRi approach as a powerful method for studying biofilm development over extended periods and revealed that numerous small non-coding RNAs are involved in regulating biofilm growth dynamics and architecture. The CRISPRi approach was also applied to a pool of 507 genes and transcription units, including protein-coding genes and non-coding RNAs, to screen for cell fitness in these two biofilm models. We discovered that, while both biofilm forms rely on fundamental processes such as cell wall synthesis and nucleotide metabolism, they exhibit different genetic dependencies with regard to matrix composition, motility, and signaling. Exopolysaccharide production, motility, and chemotaxis are crucial for pellicle formation. In contrast, macrocolony development is influenced by {gamma}-polyglutamate synthesis and nutrient acquisition functions. Genes of unknown function were also identified to play a differentially important role in the two biofilm forms. Additionally, the CRISPRi screens revealed further non-coding RNAs regulating biofilm architecture and growth dynamics, adding to the existing layers of post-transcriptional control. Collectively, these results demonstrate that biofilm formation at different physical interfaces is governed by a combination of shared and unique genetic pathways tailored to the specific biofilm environment, thereby opening research avenues into the molecular mechanisms specific to the solid-air and liquid-air interfaces.

Antibiotic resistance is a growing global health crisis, yet resistance is almost exclusively quantified under aerobic laboratory conditions that fail to reflect the complex microenvironments bacteria encounter during infection. Many clinically important infection sites, such as airways of individuals with cystic fibrosis or chronic wounds, are microaerobic to anoxic. To address this, we investigated how anoxia alters antibiotic resistance determinants, hypothesizing that anaerobic metabolism might change the fitness effects and selection of resistance mutations. We used experimental evolution to propagate Pseudomonas aeruginosa populations for approximately 200 generations under conditions differing in oxygen availability (oxic vs. anoxic), growth mode (biofilm vs. planktonic), and tobramycin (TOB) exposure (subinhibitory increasing to inhibitory concentrations). Subinhibitory exposure was sufficient to achieve resistance 2-4x greater than ancestral levels, with anoxic populations consistently showing higher minimum inhibitory concentrations than oxic comparisons. Resistance developed through condition-dependent genomic targets: mutations in amgS were selected in oxic populations, while fusA1 and ptsP mutations arose across all conditions. Notably, mexT mutations were nearly universally selected, particularly under anoxic or tobramycin-exposed conditions. mexT inactivation may also enhance virulence through altered quorum sensing and increased rhamnolipid production. Anoxic populations additionally exhibited significantly increased biofilm formation, some exceeding 1000% of ancestral levels, reduced twitching motility driven by type IV pilus gene mutations, and greater competitive fitness. Together, these findings demonstrate that oxygen availability shapes resistance evolution in P. aeruginosa, with the anoxic environment selecting for a more virulent, sessile, and antibiotic-resistant phenotype.

Gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, poses a growing global public health threat due to the rapid emergence of multidrug-resistant strains and the limited availability of effective treatments. Since there are no known lytic gonophages, we explored prophages present in the genome of N. gonorrhoeae FA1090, with a particular focus on prophage-encoded endolysins. In this study, we evaluate antigonococcal properties of prophage-encoded endopeptidases with the NlpC/P60 enzymatic domain. Recombinant endolysin Phi1gp518 exhibits intrinsic bactericidal activity against non-permeabilized N. gonorrhoeae FA1090 cells. Furthermore, it shows an expanded host range against clinical gonococcal isolates. The gonolysin remains stable across all human body temperatures, a pH range of 5-10, and shows no cytotoxic effects toward human cervical epithelial cells, supporting its potential safety for therapeutic applications. Additionally, Phi1gp518 impairs the formation of gonococcal microcolonies and prevents proper biofilm establishment. The antigonococcal properties of Phi1gp518 endopeptidase make it a good candidate for further protein engineering and development as an alternative treatment strategy for drug-resistant N. gonorrhoeae infections.

A paradigm shift is underway in microbiology: many prokaryotes, long considered to lack the compartmentalization present in all eukaryotic life, have been found to possess a great diversity of protein based intracellular compartments. Notably, the genomes of many marine and freshwater anaerobic ammonium oxidizing (anammox) bacteria encode one of these compartmentalization strategies; encapsulin nanocompartments. These systems structure suggests a role for anammox encapsulins in the anammox metabolism, a process of global biogeochemical significance, which results in the loss of biologically available nitrogen from aquatic environments. Here we test if the most common anammox encapsulin architecture could provide a mechanism to detoxify NO, one of the reactive intermediates produced in the core anammox metabolism. Through experiments in which the Kuenenia stuttgartiensis encapsulin was heterologously expressed by an inducible plasmid in E. coli, we show evidence that suggests the K. stuttgartiensis encapsulin provides no protection from NO.