CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have determined the high-resolution three-dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the single-wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg(2+)-dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.

Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation.

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.

Residual chlorine is often required to remain present in public drinking water supplies during distribution to ensure water quality. It is essential to understand how bacteria respond to long-term chlorine exposure, especially with the presence of assimilable organic carbon (AOC). This study aimed to investigate the effects of chlorination on Pseudomonas aeruginosa in low AOC medium by both conventional plating and culture-independent methods including flow cytometry (FCM) and quantitative PCR (qPCR). In a simulated chlorinated system using a bioreactor, membrane damage and DNA damage were measured by FCM fluorescence fingerprint. The results indicated membrane permeability occurred prior to DNA damage in response to chlorination. A regrowth of P. aeruginosa was observed when the free chlorine concentration was below 0.3 mg/L. The bacterial response to long-term exposure to a constant low level of free chlorine (0.3 mg/L) was subsequently studied in detail. Both FCM and qPCR data showed a substantial reduction during initial exposure (0-16 h), followed by a plateau where the cell concentration remained stable (16-76 h), until finally all bacteria were inactivated with subsequent continuous chlorine exposure (76-124 h). The results showed three-stage inactivation kinetics for P. aeruginosa at a low chlorine level with extended exposure time: an initial fast inactivation stage, a relatively stable middle stage, and a final stage with a slower rate than the initial stage. A series of antibiotic resistance tests suggested long-term exposure to low chlorine level led to the selection of antibiotic-resistant P. aeruginosa. The combined results suggest that depletion of residual chlorine in low AOC medium systems could reactivate P. aeruginosa, leading to a possible threat to drinking water safety.

NrtR is a Nudix-related transcriptional regulator that is distributed among diverse bacteria and plays an important role in modulating bacterial intracellular NAD homeostasis. Previously, we showed that NrtR influences the T3SS expression and pathogenesis of Pseudomonas aeruginosa and demonstrated that NrtR mediates T3SS regulation through the cAMP/Vfr pathway. In the present study, we found that mutation of the nrtR gene leads to upregulation of the Hcp secretion island-I type VI secretion system (H1-T6SS). Further analysis revealed that mutation of the nrtR gene results in upregulation of regulatory RNAs (RsmY/RsmZ) that are known to control the H1-T6SS by sequestration of RsmA or RsmN. Simultaneous deletion of rsmY/rsmZ reduced the expression of H1-T6SS in the ΔnrtR mutant. In addition, overexpression of either rsmA or rsmN in ΔnrtR decreased H1-T6SS expression. Chromatin immunoprecipitation (ChIP)-Seq and electrophoretic mobility shift assay (EMSA) analyses revealed that NrtR directly binds to the promoters of rsmY, rsmZ and tssA1 (first gene of the H1-T6SS operon). Overall, the results from this study reveal the molecular details of NrtR-mediated regulation of H1-T6SS in P. aeruginosa. IMPORTANCE NrtR is a Nudix-related transcriptional regulator and controls the NAD cofactor biosynthesis in bacteria. P. aeruginosa NrtR binds to the intergenic region between nadD2 and pcnA to repress the expression of the two operons, therefore controlling the NAD biosynthesis. We have previously reported that NrtR controls T3SS expression via the cAMP/Vfr pathway in P. aeruginosa. However, the global regulatory function and direct binding targets of the NrtR remain elusive in P. aeruginosa. This study reveals novel direct regulatory targets of the NrtR in P. aeruginosa, elucidating the molecular mechanism of NrtR-mediated regulation of H1-T6SS.

Pseudomonas aeruginosa is an important nosocomial pathogen which frequently becomes resistant to most antibiotics used in chemotherapy, resulting in treatment failure among infected individuals. Although the evolutionary trajectory and molecular mechanisms for becoming β-lactam resistant have been well established for P. aeruginosa, the molecular basis of reversion from β-lactam resistant to susceptible is largely unexplored. In this study, we investigated the molecular mechanisms by which a ceftazidime-resistant clinical strain is converted to a ceftazidime-susceptible isolate under the clinical setting. RNA sequencing and genomic DNA reference mapping were conducted to compare the transcriptional profiles and chromosomal mutations between these two isolates. Our results demonstrate that a gain-of-function mutation in ampD, via deletion of a 53 bp duplicated nucleotide sequence, is the contributory factor for the conversion. Furthermore, we show for the first time that AmpD is involved in intraspecies competitiveness in P. aeruginosa. We also found that AmpD is not responsible for phenotypic changes between R1 and S2, including growth rate, motilities, pyocyanin, rhamnolipid, and biofilm production. This finding provides novel insights into the alteration of β-lactam sensitivity in P. aeruginosa under the clinical setting.

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.

Pseudomonas aeruginosa is a ubiquitous pathogen that causes a wide range of acute and chronic infections. Ciprofloxacin, one of the first-line fluoroquinolone class antibiotics, is commonly used for the treatment of P. aeruginosa infections. However, ciprofloxacin-resistant P. aeruginosa is increasingly reported worldwide, making treatment difficult. To determine resistance-related mutations, we conducted an experimental evolution using a previously identified ciprofloxacin-resistant P. aeruginosa clinical isolate, CRP42. The evolved mutants could tolerate a 512-fold higher concentration of ciprofloxacin than CRP42. Genomic DNA reference mapping was performed, which revealed mutations in genes known to be associated with ciprofloxacin resistance as well as in those not previously linked to ciprofloxacin resistance, including the ParER586W substitution and PA0625 frameshift insertion. Simulation of the ParER586W substitution and PA0625 frameshift insertion by gene editing in CRP42 and the model strain PAO1 demonstrated that while the PA0625 mutation does contribute to resistance, mutation in the ParER586W does not contribute to resistance but rather affects tolerance against ciprofloxacin. These findings advance our understanding of ciprofloxacin resistance in P. aeruginosa.

YfiBNR is a tripartite signalling system in Pseudomonas aeruginosa that modulates intracellular c-di-GMP levels in response to signals received in the periplasm. YfiB is an outer membrane lipoprotein and presumed sensor protein that sequesters the repressor protein YfiR. To provide insights into YfiBNR function, we have determined three-dimensional crystal structures of YfiB and YfiR from P. aeruginosa PAO1 alone and as a 1:1 complex. A YfiB(27-168) construct is predominantly dimeric, whereas a YfiB(59-168) is monomeric, indicating that YfiB can dimerize via its N-terminal region. YfiR forms a stable complex with YfiB(59-168), while the YfiR binding interface is obstructed by the N-terminal region in YfiB(27-168). The YfiB-YfiR complex reveals a conserved interaction surface on YfiR that overlaps with residues predicted to interact with the periplasmic PAS domain of YfiN. Comparison of native and YfiR-bound structures of YfiB suggests unwinding of the N-terminal linker region for attachment to the outer membrane. A model is thus proposed for YfiR sequestration at the outer membrane by YfiB. Our work provides the first detailed insights into the interaction between YfiB and YfiR at the molecular level and is a valuable starting point for further functional and mechanistic studies of the YfiBNR signalling system.

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.

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

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 β-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 β-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.

Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins.

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.

Pseudomonas aeruginosa is a ubiquitous pathogenic bacterium that can adapt to a variety environments. The ability to effectively sense and respond to host local nutrients is critical for the infection of P. aeruginosa. However, the mechanisms employed by the bacterium to respond to nutrients remain to be explored. CspA family proteins are RNA binding proteins that are involved in gene regulation. We previously demonstrated that the P. aeruginosa CspA family protein CspC regulates the type III secretion system in response to temperature shift. In this study, we found that CspC regulates the quorum-sensing (QS) systems by repressing the translation of a QS negative regulatory gene, rsaL. Through RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) and electrophoretic mobility shift assays (EMSAs), we found that CspC binds to the 5' untranslated region of the rsaL mRNA. Unlike glucose, itaconate (a metabolite generated by macrophages during infection) reduces the acetylation of CspC, which increases the affinity between CspC and the rsaL mRNA, leading to upregulation of the QS systems. Our results revealed a novel regulatory mechanism of the QS systems in response to a host-generated metabolite. IMPORTANCE Bacterial infectious diseases impose a severe threat to human health. The ability to orchestrate virulence determinant in response to the host environment is critical for the pathogenesis of bacterial pathogens. Pseudomonas aeruginosa is a leading pathogen that causes various infections in humans. In P. aeruginosa, the quorum-sensing (QS) systems play an important role in regulating the production of virulence factors. In this study, we find that a small RNA binding protein, CspC, regulates the QS systems by repressing the expression of a QS negative regulator. We further demonstrate that CspC is acetylated in response to a host-derived metabolite, itaconate, which alters the function of CspC in regulating the QS system. The importance of this work is in elucidation of a novel regulatory pathway that regulates virulence determinants in P. aeruginosa in response to a host signal.

Polybrominated diphenyl ethers (PBDEs) have become widespread environmental pollutants all over the world. A newly isolated bacterium from an e-waste recycling area, Stenotrophomonas sp. strain WZN-1, can degrade decabromodiphenyl ether (BDE 209) effectively under aerobic conditions. Orthogonal test results showed that the optimum conditions for BDE 209 biodegradation were pH 5, 25 °C, 0.5% salinity, 150 mL minimal salt medium volume. Under the optimized condition, strain WZN-1 could degrade 55.15% of 65 μg/L BDE 209 under aerobic condition within 30 day incubation. Moreover, BDE 209 degradation kinetics was fitted to a first-order kinetics model. The biodegradation mechanism of BDE 209 by strain WZN-1 were supposed to be three possible metabolic pathways: debromination, hydroxylation, and ring opening processes. Four BDE 209 degradation genes, including one hydrolase, one dioxygenase and two dehalogenases, were identified based on the complete genome sequencing of strain WZN-1. The real-time qPCR demonstrated that the expression level of four identified genes were significantly induced by BDE 209, and they played an important role in the degradation process. This study is the first to demonstrate that the newly isolated Stenotrophomonas strain has an efficient BDE 209 degradation ability and would provide new insights for the microbial degradation of PBDEs.

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.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5' untranslated region (5' UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5' UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

Betaproteobacterium strain CB is a typical minute freshwater bacterium, representing the small-cell bacteria that are numerically dominant in most freshwater environments. The genome of betaproteobacterium CB consists of a circular 2,045,720-bp chromosome, and the information we report will provide insights into the mechanisms underlying its survival and ecological function.