Polystyrene nanoparticles induce biofilm formation in Pseudomonas aeruginosa.

In recent years, micro/nanoplastics have garnered widespread attention due to their ecological risks. In this study, we investigated the effects of polystyrene nanoparticles (PS-NPs) of different sizes on the growth and biofilm formation of Pseudomonas aeruginosa PAO1. The results demonstrated that exposure to certain concentrations of PS-NPs significantly promoted bacterial biofilm formation. Meanwhile, we comprehensively revealed its mechanism whereby PS-NPs induced oxidative stress and altered bacterial membrane permeability by contacting or penetrating bacterial membranes. To counteract the stimulation by PS-NPs and reduce their toxicity, bacteria enhanced biofilm formation by upregulating the expression of biofilm-related genes, increasing EPS and virulence factors secretion, and enhancing bacterial motility through the participation of the quorum sensing (QS) system. Additionally, we also found that exposure to PS-NPs enhanced bacterial antibiotic resistance, posing a challenge to antimicrobial therapy. Our study reveals the toxic effects of nanoplastics and the defense mechanisms of bacteria, which has important implications for the risk assessment and management of environmental nanoplastics.

Structure and function of the pyridoxal 5'-phosphate-dependent (PLP) threonine deaminase IlvA1 from Pseudomonas aeruginosa PAO1.

IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.

Low and high nucleic acid content bacteria play discrepant roles in response to various carbon supply modes.

Planktonic bacteria can be grouped into 'high nucleic acid content (HNA) bacteria' and 'low nucleic acid content (LNA) bacteria.' Nutrient input modes vary in environments, causing nutrient availability heterogeneity. We incubated them with equal amounts of total glucose added in a continuous/pulsed mode. The pulse-treated LNA bacteria exhibited twice the cell abundance and four times the viability of the continuous-treated LNA, while HNA did not show an adaptation to pulsed treatment. In structural equation modelling, LNA bacteria had higher path coefficients than HNA, between growth and carbon-saving metabolic pathways, intracellular ATP and the inorganic energy storage polymer, polyphosphate, indicating their low-cost growth, and flexible energy storage and utilisation. After incubation, the pulse-treated LNA bacteria contained more proteins and polysaccharides (0.00064, 0.0012 ng cell-1 ) than the continuous-treated LNA (0.00014, 0.00014 ng cell-1 ), conferring endurance and rapid response to pulses. Compared to LNA, HNA keystone taxa had stronger correlations with the primary glucose metabolism step, glycolysis, and occupied leading positions to explain the random forest model. They are essential to introduce glucose into the element cycling of the whole community under both treatments. Our work outlines a systematic bacterial response to carbon input.

Structure and function of molecular machines involved in deadenylation-dependent 5'-3' mRNA degradation.

In eukaryotic cells, the synthesis, processing, and degradation of mRNA are important processes required for the accurate execution of gene expression programmes. Fully processed cytoplasmic mRNA is characterised by the presence of a 5'cap structure and 3'poly(A) tail. These elements promote translation and prevent non-specific degradation. Degradation via the deadenylation-dependent 5'-3' degradation pathway can be induced by trans-acting factors binding the mRNA, such as RNA-binding proteins recognising sequence elements and the miRNA-induced repression complex. These factors recruit the core mRNA degradation machinery that carries out the following steps: i) shortening of the poly(A) tail by the Ccr4-Not and Pan2-Pan3 poly (A)-specific nucleases (deadenylases); ii) removal of the 5'cap structure by the Dcp1-Dcp2 decapping complex that is recruited by the Lsm1-7-Pat1 complex; and iii) degradation of the mRNA body by the 5'-3' exoribonuclease Xrn1. In this review, the biochemical function of the nucleases and accessory proteins involved in deadenylation-dependent mRNA degradation will be reviewed with a particular focus on structural aspects of the proteins and enzymes involved.

Tea polyphenols inhibit blooms caused by eukaryotic and prokaryotic algae.

With changes in global climate, blooms are becoming more frequent and difficult to control. Therefore, the selection of algal suppressor agents with effective inhibition and environmental safety is of paramount importance. One of the main treatment strategies is to inhibit the release of harmful algal toxins. Tea polyphenols (TP) are natural products that have been widely used in medicine, the environment, and other fields due to their antibacterial and antioxidant properties. To investigate their potential application in the treatment of algal blooms, TP were applied to three different microalgae. TP exhibited strong inhibitory effects towards all three microalgae. They stimulate the accumulation of ROS in algal cells, leading to lipid peroxidation and subsequent damage to the cell membrane, resulting in the rupture and necrosis of Cyclotella sp. and Chlorella vulgaris cells. Remarkably, it was observed that lower concentrations of TP exhibited the ability to induce apoptosis in M. aeruginosa cells without causing any structural damage. This outcome is particularly significant as it reduces the potential risk of microcystin release resulting from cell rupture. Overall, blooms dominated by different algae can be treated by adjusting the concentration of TP, a new algal suppressor, indicating strong potential treatment applications.

Structural analysis of the ferric-binding protein KfuA from Klebsiella pneumoniae.

Iron acquisition is an essential process of cell physiology for biological systems. In Klebsiella pneumoniae, the siderophore and ferric-acquisition ABC (ATP-Binding-Cassette) transporter KfuABC is utilized for iron uptake. Initial recognition of the various ferric sources in periplasm and transportation across the cytoplasmic membrane is performed by the substrate-binding protein (SBP) KfuA. Here we report the 2.0 Å resolution crystal structure of KfuA from K. pneumoniae, which crystallizes in the space group P1211 with a single monomer in the asymmetric unit. A bound metal ion reveals the residues required for binding ferric ions. Binding analysis shows that ferric iron and the iron-mimicking gallium bind with high affinity to KfuA. Growth curves show that gallium inhibits growth of K. pneumoniae whereas ferric iron enhances it. This work suggests a mechanism whereby gallium effectively competes with ferric iron, disrupting iron-dependent biological functions via binding to KfuA and leading to heightened antimicrobial efficacy. Significantly, humans lack equivalent ABC transporters like SBP KfuA, underscoring the potential of KfuA as an attractive target for therapeutic intervention.

Influence of phycospheric bacterioplankton disruption or removal on algae growth and survival.

Phycospheric bacteria play a crucial role in the survival of microalgae. However, the potential of using the growth regulation and community structure modulation of phycospheric bacteria to prevent the occurrence of blooms is yet to be verified. The phycospheric bacterioplankton of Cyclotella sp. can be categorized into HNA (high nucleic acid) bacteria and LNA (low nucleic acid) bacteria. 16S rRNA sequencing showed that the HNA bacteria exhibited higher α-diversity compared to the LNA bacteria, and the microbial community composition also exhibited variations. Metagenomic sequencing further indicated the distinct ecological functions between HNA and LNA bacteria. Furthermore, the study showcased the restorative capacity of the phycospheric bacterioplankton. Biomass analysis revealed that the recovery of phycospheric bacterioplankton positively influenced the microalgae growth, thus affirming the significance of phycospheric bacterioplankton to microalgae. The community structure of phycospheric bacterioplankton demonstrated a notable decrease in the abundance of restored LNA core bacteria. Additionally, the restored phycospheric bacterioplankton exhibited a more complex co-occurrence network structure, resulting in decreased resistance and sensitivity of microalgae to adverse environments. The presence of phycospheric bacterioplankton provides a protective shield for microalgae, and thus destabilizing or removing phycospheric bacterioplankton may effectively inhibit growth of microalgae.

Structural analysis of molybdate binding protein ModA from Klebsiella pneumoniae.

Klebsiella pneumoniae, a facultative anaerobe, relies on acquiring molybdenum to sustain growth in anaerobic conditions, a crucial factor for the pathogen to establish infections within host environments. Molybdenum plays a critical role in pathogenesis as it forms an essential component of cofactors for molybdoenzymes. K. pneumoniae utilizes the ABC (ATP-Binding-Cassette) transporter encoded by the modABC operon for uptake of the group VI elements molybdenum and tungsten. In this study, we determined the X-ray crystal structures of both the molybdenum-free and molybdenum-bound substrate-binding protein (SBP) ModA from Klebsiella pneumoniae to 2.00 Å and 1.77 Å resolution respectively. ModA crystallizes in the space group P222 with a single monomer in one asymmetric unit. The purified protein remained soluble and specifically bound molybdate and tungstate with Kd values of 6.3 nM and 5.2 nM, respectively. Tungstate competes with molybdate by binding to ModA, resulting in enhanced antimicrobial activity. These data provide a starting point for structural and functional analyses of molybdate transport in K. pneumoniae.

Spatiotemporal dynamics of high and low nucleic acid-content bacterial communities in Chinese coastal seawater: assembly process, co-occurrence relationship and the ecological functions.

Studies of high nucleic acid-content (HNA) and low nucleic acid-content (LNA) bacterial communities are updating our view of their distributions and taxonomic composition. However, there are still large gaps in our knowledge of the composition, assembly processes, co-occurrence relationships and ecological functions of HNA and LNA bacterial communities. Here, using 16S rRNA gene amplicon sequencing, we investigated the spatiotemporal dynamics, assembly processes, co-occurrence relationships and ecological functions of HNA and LNA bacterial communities in the samples collected in summer and winter in Chinese coastal seas. The communities of HNA and LNA bacteria had clear spatiotemporal patterns and LNA bacteria was phylogenetically less diverse than HNA bacteria in both seasons. The distribution of HNA and LNA bacteria were significantly affected by the environmental factors and a significant seasonal-consistent distance-decay patterns were found in HNA and LNA bacteria. Furthermore, a quantitative assessment of ecological processes revealed that dispersal limitation, homogeneous selection exerted important roles in the community assembly of HNA and LNA bacteria. More importantly, we observed seasonality in the co-occurrence relationships: closer inter-taxa connections of HNA bacterial communities in winter than in summer and the opposite is true in the LNA bacterial communities. Some ecological functions, such as: phototrophy, photoautotrophy, oxygenic photoautotrophy, were different between HNA and LNA bacteria. These results provide a better understanding of spatiotemporal patterns, processes, and the ecological functions of HNA and LNA bacterial communities in Chinese coastal seawater.

Anthropogenic original DOM is a critical factor affecting LNA bacterial community assembly.

We investigated the geographical and environmental distance-decay relationships for both of the two bacteria in the Haihe River, Tianjin, China. HNA bacteria exhibited a stronger geographical variation-dependent pattern while LNA bacteria exhibited a stronger environmental variation-dependent pattern. Variance partition analysis (VPA), Mantel test, and partial mantel test validated the discrepant impacts of geographical distance and environmental factors on their two communities. The heterogeneous selection dominated community assembly of LNA bacteria demonstrates their greater sensitivity to environmental conditions. As the deterministic environmental factor, anthropogenic original dissolved organic matter (DOM) functions exclusively on LNA bacteria, and it is the critical factor leading to the discrepant biogeographical patterns of LNA and HNA bacteria. LNA bacteria interact with HNA bacteria and mediate the DOM driving total bacteria assembly. The LNA keystone taxa, Pseudomonas, Rheinheimera, Candidatus Aquiluna, and hgcl clade are capable to compete with HNA bacteria for anthropogenic original DOM, and are potential indicators of anthropogenic pollution. Our research reveals the non-negligible effect of the LNA bacteria in regulating the ecological response of total bacteria.

Regulatory and structural mechanisms of PvrA-mediated regulation of the PQS quorum-sensing system and PHA biosynthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is capable of causing acute and chronic infections in various host tissues, which depends on its abilities to effectively utilize host-derived nutrients and produce protein virulence factors and toxic compounds. However, the regulatory mechanisms that direct metabolic intermediates towards production of toxic compounds are poorly understood. We previously identified a regulatory protein PvrA that controls genes involved in fatty acid catabolism by binding to palmitoyl-coenzyme A (CoA). In this study, transcriptomic analyses revealed that PvrA activates the Pseudomonas quinolone signal (PQS) synthesis genes, while suppressing genes for production of polyhydroxyalkanoates (PHAs). When palmitic acid was the sole carbon source, mutation of pvrA reduced production of pyocyanin and rhamnolipids due to defective PQS synthesis, but increased PHA production. We further solved the co-crystal structure of PvrA with palmitoyl-CoA and identified palmitoyl-CoA-binding residues. By using pvrA mutants, we verified the roles of the key palmitoyl-CoA-binding residues in gene regulation in response to palmitic acid. Since the PQS signal molecules, rhamnolipids and PHA synthesis pathways are interconnected by common metabolic intermediates, our results revealed a regulatory mechanism that directs carbon flux from carbon/energy storage to virulence factor production, which might be crucial for the pathogenesis.

Structural characterization of the novel stress response facilitator (SrfA) from Pseudomonas aeruginosa.

Chronic pulmonary infections in those living with cystic fibrosis or chronic obstructive pulmonary disease are promoted by production of alginate by the opportunistic pathogen Pseudomonas aeruginosa. Alginate biosynthesis enzymes in P. aeruginosa are regulated by the extracytoplasmic function alternative sigma factor σ22 either by mutation in mucA or in response to envelope stress. An intergenic region between ORFs PA2559 and PA2560 in P. aeruginosa is σ22-dependent and its transcription is activated by cell wall stress. This stress-responsive transcript encodes a novel stress response facilitator, SrfA, that is exclusively conserved only in P. aeruginosa species. Here we report the first three-dimensional structure of SrfA determined by molecular replacement using fold prediction to generate a search model. The SrfA structure adopts a helix-loop-helix fold that shares some similarity with structures of anti-activator or effector proteins. A ΔsrfA mutant strain of P. aeruginosa PAO1 exhibited significantly reduced biofilm formation, which was restored to wild-type levels when ΔsrfA was complemented with srfA. The ΔsrfA strain also exhibited increased sensitivity to macrolide antibiotics. We further show using MicroScale Thermophoresis that SrfA interacts with both PA2559 and PA2560 with high affinity. This work provides a starting point for further investigation into the role of SrfA in response to cell wall stress.

Transcriptome Profiling of Stenotrophomonas sp. Strain WZN-1 Reveals Mechanisms of 2,2',4,4'-Tetrabromodiphenyl Ether (BDE-47) Biotransformation.

The brominated flame retardant 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is extensively used, stable, and difficult to degrade in the environment. The existence of BDE-47 could pose a certain risk to the environment and human health. However, the biotransformation mechanisms of BDE-47 by microorganisms remain unclear. In this study, aerobic degradation of BDE-47 by Stenotrophomonas sp. strain WZN-1 and transcriptome analysis were carried out. BDE-47 degradation by Stenotrophomonas sp. strain WZN-1 was mainly through the biological action of intracellular enzymes via the route of debromination and hydroxylation. The results of the transcriptome sequencing indicated the differentially expressed genes were related to transport, metabolism, and stress response. The key processes involved the microbial transmembrane transportation of BDE-47, energy anabolism, synthesis, and metabolism of functional enzymes, stress response, and other biological processes of gene regulation. In particular, bacterial chemotaxis played a potential role in biodegradation of BDE-47 by Stenotrophomonas sp. strain WZN-1. This study provides the first insights into the biotransformation of Stenotrophomonas sp. strain WZN-1 to BED-47 stress and shows potential for application in remediation of polluted environments.

Characteristics, Biodiversity, and Cultivation Strategy of Low Nucleic Acid Content Bacteria.

Low nucleic acid content (LNA) bacteria are ubiquitous and estimated to constitute 20%-90% of the total bacterial community in marine and freshwater environment. LNA bacteria with unique physiological characteristics, including small cell size and small genomes, can pass through 0.45-µm filtration. The researchers came up with different terminologies for low nucleic acid content bacteria based on different research backgrounds, such as: filterable bacteria, oligotrophic bacteria, and low-DNA bacteria. LNA bacteria have an extremely high level of genetic diversity and play an important role in material circulation in oligotrophic environment. However, the majority of LNA bacteria in the environment remain uncultivated. Thus, an important challenge now is to isolate more LNA bacteria from oligotrophic environments and gain insights into their unique metabolic mechanisms and ecological functions. Here, we reviewed LNA bacteria in aquatic environments, focusing on their characteristics, community structure and diversity, functions, and cultivation strategies. Exciting future prospects for LNA bacteria are also discussed.

Structural characterization of an L-fuculose-1-phosphate aldolase from Klebsiella pneumoniae.

Fuculose phosphate aldolases play an important role in glycolysis and gluconeogenesis pathways. L-fuculose 1-phosphate aldolase catalyzes the reversible cleavage of L-fuculose 1-phosphate to DHAP and L-lactaldehyde. Class II aldolases found in bacteria are linked to pathogenesis of human pathogens, and have potential applications in the biosynthesis of carbohydrates and other chiral compounds. Here we report the structure of a putative L-fuculose 1-phosphate aldolase (KpFucA) from the nosocomial pathogen Klebsiella pneumoniae to 1.85 Å resolution. The enzyme crystallizes in space group P422 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis confirms that KpFucA is a tetramer in solution. A magnesium ion cofactor and sulfate ion were identified in the active pocket. Enzyme activity assays confirmed that KpFcuA has a strong preference for L-fuculose 1-phosphate as a substrate, but can also catalyze the cleavage of fructose-1,6-bisphosphate and glucose-6-phosphate. This work should provide a starting point for further investigation of the role of KpFucA in K. pneumoniae pathogenesis or in industrial applications.

Structural characterization of the urease accessory protein UreF from Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that mostly affects those with weakened immune systems. Urease is a vital enzyme that can hydrolyze urea to ammonia and carbon dioxide as a source of nitrogen for growth. Urease is also a K. pneumoniae virulence factor that enables survival of the bacterium under nutrient-limiting conditions. UreF, an important nickel-binding urease accessory protein, is involved in the insertion of Ni2+ into the active site of urease. Here, the crystal structure of UreF from K. pneumoniae (KpUreF) is reported. Functional data show that KpUreF forms a stable dimer in solution. These results may provide a starting point for the design of urease inhibitors.

Integrated metagenomic and metatranscriptomic analysis reveals actively expressed antibiotic resistomes in the plastisphere.

The plastisphere is viewed as a reservoir for the antibiotic resistome in water environments and may pose health concerns. However, the expression profiles of the resistome in the plastisphere are largely unknown. Here, we profiled the occurrence, abundance, and transcriptional level of antibiotic resistance genes (ARGs), plasmid associated ARGs, microbial composition and ARG bacterial hosts in the plastisphere and urban river water using 16S rRNA gene sequencing, metagenomic sequencing, and metatranscriptomic sequencing methods. A total of 173 ARGs conferring resistance to 24 major classes of antibiotics commonly prescribed to humans and animals were detected in the plastisphere. Of these, 75 genes were observed with transcriptional activity, indicating that the antibiotic resistome in the plastisphere was not only present, but also actively expressed. Human pathogens belonging to family Enterobacteriaceae were identified as bacterial hosts of ARGs in the plastisphere. The opportunistic and multidrug resistant human pathogen Enterobacter cloacae was found to actively express tetG and confer tetracycline resistance to the plastisphere. Furthermore, 39 genes were identified as "plasmid associated ARGs" in the plastisphere, displaying a higher proportion of transcript abundance compared with water. The above results suggest that the plastisphere is a hotspot for antibiotic resistome acquisition, expression, and dissemination.

Structure of the human Ccr4-Not nuclease module using X-ray crystallography and electron paramagnetic resonance spectroscopy distance measurements.

Regulated degradation of mature, cytoplasmic mRNA is a key step in eukaryotic gene regulation. This process is typically initiated by the recruitment of deadenylase enzymes by cis-acting elements in the 3' untranslated region resulting in the shortening and removal of the 3' poly(A) tail of the target mRNA. The Ccr4-Not complex, a major eukaryotic deadenylase, contains two exoribonuclease subunits with selectivity toward poly(A): Caf1 and Ccr4. The Caf1 deadenylase subunit binds the MIF4G domain of the large subunit CNOT1 (Not1) that is the scaffold of the complex. The Ccr4 nuclease is connected to the complex via its leucine-rich repeat (LRR) domain, which binds Caf1, whereas the catalytic activity of Ccr4 is provided by its EEP domain. While the relative positions of the MIF4G domain of CNOT1, the Caf1 subunit, and the LRR domain of Ccr4 are clearly defined in current models, the position of the EEP nuclease domain of Ccr4 is ambiguous. Here, we use X-ray crystallography, the AlphaFold resource of predicted protein structures, and pulse electron paramagnetic resonance spectroscopy to determine and validate the position of the EEP nuclease domain of Ccr4 resulting in an improved model of the human Ccr4-Not nuclease module.

Structure of Pseudomonas aeruginosa spermidine dehydrogenase: a polyamine oxidase with a novel heme-binding fold.

The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4-aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P. aeruginosa, but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P. aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin-binding and substrate-binding domains. A unique N-terminal extension wraps around the flavin-binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine-water-flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P. aeruginosa strains, in polyamine metabolism.

Crystal structure of oligoribonuclease from Vibrio cholerae O1 El Tor with bound peptide.

Oligoribonuclease (Orn), a member of the DEDDh superfamily, can hydrolyse 2-5 nt nanoRNAs to mononucleotides. It is involved in maintaining the intracellular levels of RNA, c-di-GMP signalling and transcription initiation in many bacterial species. Here, the crystal structure of Orn from Vibrio cholerae O1 El Tor (VcOrn) is reported at a resolution of 1.7 Å. VcOrn, which consists of nine α-helices and six ß-strands, crystallizes with a single monomer in the asymmetric unit but forms a homodimer via crystallographic twofold symmetry. Electron density is observed in the active pocket that corresponds to an intersubunit N-terminal expression tag with sequence GPLGSHHH. The positively charged N-terminal tag binds in the negatively charged nucleotide-binding pocket with a buried surface area of ∼500 Å2. The N-terminal tag interacts with VcOrn via π-π stacking with two conserved residues involved in nucleotide binding, as well as via salt bridges and hydrogen bonds. The structure reported here reveals that the active pocket can accommodate polypeptides in addition to nucleotides, thus providing an important starting point for investigation into substrate modification and inhibitor design targeting VcOrn.