The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in B. abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.

The phytopathogen Pseudomonas syringae employs an active type VI secretion system (T6SS) to outcompete other microorganisms in the natural environment, particularly during the epiphytic growth in the phyllosphere. By examining two T6SS clusters in P. syringae MB03, T6SS-1 is found to be effective in killing Escherichia coli cells. We highlight the excellent antibacterial effect of two T6SS DNase effectors, namely, Tde1 and Tde4. Both of them function as nuclease effectors, leading to DNA degradation and cell filamentation in prey cells, ultimately resulting in cell death. Our findings deepen our understanding of the T6SS effector repertoires used in P. syringae and will facilitate the development of effective antibacterial strategies.

Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.

A new study by Nies et al. (J Bacteriol 206:e00080-24, 2024, https://doi.org/10.1128/jb.00080-24) provides a rich, quantitative data set of zinc accumulation by cells of Cupriavidus metallidurans, including of mutant bacterial strains lacking import or efflux genes, and comparison of zinc accumulation by cells previously starved of metal with those of zinc-replete cells. The data surprisingly demonstrate the concomitant activity of both active metal import and metal efflux systems. They present a flow equilibrium model to describe zinc homeostasis in bacteria.

Consequences of reduction in photosystem I content were investigated in the cyanobacterium Synechocystis sp. PCC 6803 where photosystem I far exceeds the number of photosystem II complexes. Strains with less photosystem I displayed less cyclic electron transport, grew more slowly at lower light intensity and needed more light for saturation but were surprisingly normal in their whole-chain electron transport rates, implying that a significant fraction of photosystem I is dispensable for linear electron transport in cyanobacteria. These strains with reduced photosystem I levels may have biotechnological relevance as they grow well at higher light intensities.

Vibrio (Aliivibrio) fischeri's initial rise to fame derived from its alluring production of blue-green light. Subsequent studies to probe the mechanisms underlying this bioluminescence helped the field discover the phenomenon now known as quorum sensing. Orthologs of quorum-sensing regulators (i.e., LuxR and LuxI) originally identified in V. fischeri were subsequently uncovered in a plethora of bacterial species, and analogous pathways were found in yet others. Over the past three decades, the study of this microbe has greatly expanded to probe the unique role of V. fischeri as the exclusive symbiont of the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Buoyed by this optically amenable host and by persistent and insightful researchers who have applied novel and cross-disciplinary approaches, V. fischeri has developed into a robust model for microbe-host associations. It has contributed to our understanding of how bacteria experience and respond to specific, often fluxing environmental conditions and the mechanisms by which bacteria impact the development of their host. It has also deepened our understanding of numerous microbial processes such as motility and chemotaxis, biofilm formation and dispersal, and bacterial competition, and of the relevance of specific bacterial genes in the context of colonizing an animal host. Parallels in these processes between this symbiont and bacteria studied as pathogens are readily apparent, demonstrating functional conservation across diverse associations and permitting a reinterpretation of "pathogenesis." Collectively, these advances built a foundation for microbiome studies and have positioned V. fischeri to continue to expand the frontiers of our understanding of the microbial world inside animals.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Understanding the biochemical action of a single enzyme or transport protein is the pre-requisite to obtain insight into its cellular function but this is only one half of the coin. The other side concerns the question of how central metabolic functions of a cell emerge from the interplay of different proteins and other macromolecules. This paper demonstrates that a flow equilibrium of zinc uptake and efflux reactions is at the core of cellular zinc homeostasis and identifies the most important contributors to this flow equilibrium: the uptake and efflux systems and metal-binding components of the cytoplasm.

Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.