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.

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.

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.

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.

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.

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 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 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.

Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport.

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential γ-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid both in vitro and when overexpressed in E. coli Additionally, transport was greatly reduced in the presence of ß-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport ß-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.

Structural characterization of a 2-aminoethylphosphonate:pyruvate aminotransferase from Pseudomonas aeruginosa PAO1.

2-aminoethylphosphonate:pyruvate aminotransferase (AEPT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that mediates the first step in the AEP degradation pathway. It catalyzes the transamination of 2-aminoethylphosphonate (AEP) with pyruvate to phosphonoacetaldehyde and l-alanine respectively. Although the enzyme is widely present in microorganisms, there are few reports on the structure and function of AEPT to date. Here we report the crystal structure of AEPT from Pseudomonas aeruginosa PAO1 (PaAEPT) to 2.35 Å resolution in the absence of the PLP cofactor. PaAEPT crystallizes in space group P21212 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis shows that PaAEPT forms a stable dimer in solution. Our work provides a valuable starting point for further functional and mechanistic studies of the AEP degradation pathway.

Structural characterization and Kemp eliminase activity of the Mycobacterium smegmatis Ketosteroid Isomerase.

The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of interest to researchers since its discovery. Because this reaction does not exist in all currently known metabolic pathways, the computational design of Kemp eliminases has provided valuable insights into principles of enzymatic catalysis. However, it was discovered that the naturally occurring promiscuous enzymes ydbC, xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) from Mycobacterium smegmatis MC2 155. MsKSI crystallizes in the P212121 space group with two molecules in an asymmetric unit, and ultracentrifugation data confirms that it forms a stable dimer in solution, consistent with the 1.9 Å-resolution structure. Our assays confirm that MsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of the MsKSI-5NBI complex reveals that the substrate 5NBI is bound in the active pocket of the enzyme composed of hydrophobic residues. In addition, the Glu127 residue is proposed to play an important role as a general base in proton transfer and breaking weak O-N bonds to open the five-membered ring. This work provides a starting point for exploring the artificial modification of MsKSI using the natural enzyme as the backbone.

Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

Structural characterization of the Pseudomonas aeruginosa dehydrogenase AtuB involved in citronellol and geraniol catabolism.

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 µM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB.

Structural characterization of an isopenicillin N synthase family oxygenase from Pseudomonas aeruginosa PAO1.

Isopenicillin N synthase (IPNS) is a nonheme-Fe2+-dependent enzyme that mediates a key step in penicillin biosynthesis. It catalyses the conversion of the tripeptide δ-(l-α-aminoadipoyl)-l-cysteine-d-valine (ACV) to isopenicillin N, which is a key precursor to ß-lactam antibiotics. The pa4191 gene in Pseudomonas aeruginosa PAO1 has provisionally been annotated as a member of the IPNS family. In this work, we report the crystal structure of PA4191 from P. aeruginosa (PaIPNS hereafter). The 1.65 Šresolution PaIPNS structure forms a jelly roll fold and is confirmed to be a member of the IPNS family based on structural homology. A metal centre within the jelly roll consists of the strictly conserved His201, Asp203 and His257 residues. MicroScale Thermophoresis binding analysis confirms that PaIPNS is a metal-binding protein with a strong preference for iron, but that it does not bind the tripeptide ACV. Structural comparison of PaIPNS with a previously reported IPNS-ACV complex structure reveals a restricted binding pocket that is unable to accommodate ACV.

Structural characterization of an acetolactate decarboxylase from Klebsiella pneumoniae.

Acetolactate decarboxylase (ALDC) is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin), an important physiological metabolite excreted by microbes. Although the enzyme is widely present in microorganisms, few atomic structures and functions of ALDC have been reported to date. Here we report the crystal structure of ALDC from Klebsiella pneumoniae KP (KpALDC). KpALDC crystallizes in space group P3121 with one monomer per asymmetric unit. Analytical ultracentrifugation data shows that KpALDC forms a stable dimer but can exist as a tetramer in solution. A Zn2+ ion is coordinated by three strictly-conserved histidines (His198, His200 and His211) and a conserved glutamate (Glu69), but the C-terminal tail that forms part of the active site in ALDC enzymes is disordered. A complex structure with ethane-1,2-diol shows a unusual mode of binding, whereby the ligand does not coordinate the Zn2+ ion. MicroScale Thermophoresis analysis shows that KpALDC binds Zn2+ ions, but no binding of Mg2+, Ca2+ and Mn2+ ions was detected.

Structural and Functional Changes of Groundwater Bacterial Community During Temperature and pH Disturbances.

In this study, we report the characteristics of a microbial community in sampled groundwater and elucidate the effects of temperature and pH disturbances on bacterial structure and nitrogen-cycling functions. The predominant phyla of candidate OD1, candidate OP3, and Proteobacteria represented more than half of the total bacteria, which clearly manifested as a "low nucleic acid content (LNA) bacteria majority" type via flow cytometric fingerprint. The results showed that LNA bacteria were more tolerant to rapid changes in temperature and pH, compared to high nucleic acid content (HNA) bacteria. A continuous temperature increase test demonstrated that the LNA bacterial group was less competitive than the HNA bacterial group in terms of maintaining their cell intactness and growth potential. In contrast, the percentage of intact LNA bacteria was maintained at nearly 70% with pH decrease, despite a 50% decrease in total intact cells. Next-generation sequencing results revealed strong resistance and growth potential of phylum Proteobacteria when the temperature increased or the pH decreased in groundwater, especially for subclasses α-, ß-, and γ-Proteobacteria. In addition, relative abundance of nitrogen-related functional genes by qPCR showed no difference in nitrifiers or denitrifiers within 0.45 µm-captured and 0.45 µm-filterable bacteria due to phylogenetic diversity. One exception was the monophyletic anammox bacteria that belong to the phylum Planctomycetes, which were mostly captured on a 0.45-µm filter. Furthermore, we showed that both temperature increase and pH decrease could enhance the denitrification potential, whereas the nitrification and anammox potentials were weakened.

Biogeography and Diversity of Freshwater Bacteria on a River Catchment Scale.

To illustrate how freshwater bacterial community changes with geographic gradient, we investigated the spatial changes of bacterial abundance and community structures from over 200 samples on a catchment scale in the Songhua River using heterotrophic plate counts, flow cytometry, denaturing gradient gel electrophoresis, and pyrosequencing analysis. The results showed that the mainstream had higher cultivable bacteria and total bacterial concentration than tributaries in the Songhua River catchment. Response model analysis demonstrated that the bacterial community exhibits a biogeographical signature even in an interconnected river network system, and the total bacterial concentration and biodiversity were significantly correlated to latitude (p < 0.001) and longitude (p < 0.001). Multivariate redundancy analysis indicated that temperature was the most important factor driving bacterial community structure in the Songhua River, which accounts for 35.30% variance of communities, then dissolved oxygen (17.60%), latitude (17.60%), longitude (11.80%), and pH (5.88%). High-throughput pyrosequencing revealed that at the phylum level, Proteobacteria was numerically dominant (89.6%) in river catchment, followed by Bacteroidetes (8.1%) and Cyanobacteria (1.2%). The overall results revealed that the bacterial community was driven by geographical distance regardless of the continuum of the river on a catchment scale.

Structural analysis of activating mutants of YfiB from Pseudomonas aeruginosa PAO1.

Bacterial cyclic-di-GMP (c-di-GMP) is an important messenger molecule that influences diverse cellular processes including motility, virulence and cytotoxicity systems, polysaccharide synthesis and biofilm formation. The YfiBNR tripartite signalling system in P. aeruginosa modulates the cellular c-di-GMP levels in response to signals received from the periplasm. In this study, we analyse the structures of activating mutants of the outer membrane protein YfiB that give rise to increased surface attachment and biofilm formation. The F48S and W55L mutants of YfiB(27-168) crystallize in the same dimeric arrangement as our previously reported YfiB structures that preclude complex formation with YfiR. The L43P mutant of YfiB(27-168) is monomeric and forms a stable complex with YfiR. The YfiB(L43P)-YfiR crystal structure reveals a dramatic rearrangement of the N-terminal fragment, which is implicated in increased YfiB activation and membrane attachment, upon YfiR binding. Comparison with our previous complex structure between YfiB(59-168) and YfiR reveals extensive interactions between the N-terminal fragment of YfiB (residues 35-55) and YfiR.