In Pseudomonas aeruginosa, SadB acts as a post-translational adaptor protein that binds to the transcriptional regulator AmrZ. Deletion of sadB results in a biofilm-defective, hyperswarming phenotype. To investigate whether SadB contributes to virulence, we employed in vivo bioluminescence imaging and histopathology to visualize the development of infection in a mouse soft tissue model. Compared with the parent PAO1 strain, the sadB mutant was highly attenuated and rapidly cleared from the infection site, whereas genetic complementation conferring constitutive expression of sadB resulted in a much more persistent phenotype. Transcriptome analysis was undertaken to gain insights into the global impact of SadB, revealing that it modulates expression of diverse genes involved in biofilm development, quorum sensing (QS), secondary metabolite production, iron acquisition, virulence, protein secretion, and anaerobiosis. In the sadB mutant, we observed log-phase induction of the rhl and pqs QS systems, increased production of siderophores and pyocyanin, differential regulation of genes involved in c-di-GMP signaling, and a growth defect under microaerophilic conditions. The overproduction of rhamnolipids is consistent with the hyperswarming, biofilm-defective phenotype of the sadB mutant since rhamnolipids act as anti-adhesive surface lubricants. Deletion of rhlA in the sadB mutant resulted in the restoration of biofilm formation, offering mechanistic insight into the biofilm-defective phenotype of the sadB mutant. SadB clearly plays a global role in the adaptive behavior and virulence of P. aeruginosa.IMPORTANCEBiofilms are characterized by their intrinsic tolerance to antibiotics, host immune defenses, and ability to cause persistent infections. In Pseudomonas aeruginosa, mutation of the surface attachment defect gene, sadB, results in cells that are biofilm-defective, hyperswarmers. Here, we sought to determine whether SadB regulates virulence and influences the development of infection. In a mouse skin infection model, a P. aeruginosa sadB deletion mutant was highly attenuated. We also demonstrate that SadB regulates many different genes involved in virulence, quorum sensing, iron acquisition, protein secretion, and anaerobiosis as well as biofilm formation, highlighting a broader role in pathogenesis than previously recognized. Consequently, SadB has potential as a novel protein target for antibacterial drug discovery.
Insights into transition metal homeostasis of Cupriavidus metallidurans have relied heavily on the construction and characterization of deletion mutants. To reveal the genetic consequences of these deletions, the genomes of C. metallidurans strain CH34 wild type and 34 of its mutants were analyzed. The genome of C. metallidurans wild type remained stable when the strain was strictly kept under the appropriate conditions. Omission of selection pressure or construction of mutants, however, resulted in three different kinds of mutations. Large deletions affected genomic islands or regions in the vicinity of transposon-associated genes. Some of these large deletions could be assigned to groups of overlapping deletions associated with groups of mutants, for instance, those with deletions in metal-efflux systems. Second, single-nucleotide polymorphisms (SNPs), such as point mutations, small insertions, or deletions, with a high variant frequency, were candidates for suppressor mutations, for example, in the corA1 or yidC genes. The third and novel kind of mutations comprised hypervariable regions, groups of SNPs with low variant frequencies in a small region of up to 70 base pairs that were located upstream or within genes. In the well-studied example involving the central zinc uptake regulator Zur, the hypervariable regions in the zur gene enabled the production of a GTP cyclohydrolase, restoring folate biosynthesis in double mutants. These data demonstrate that mutations de-stabilize the genome of C. metallidurans, accumulating changes that result in adaptation to the absence of the respective deleted gene, in some cases via hypervariable regions.IMPORTANCETo determine how bacteria thrive, they are often isolated from their natural environment and maintained in a laboratory. Mutations are introduced, and these derivatives are characterized phenotypically. Understanding how bacteria and their derivatives adapt to the effects of site-directed mutations, curing of plasmids, or just the laboratory environment is important. We show here that changes in the genomes of Cupriavidus metallidurans mutants occurred in all instances except when the wild type was maintained under selection conditions. The secondary mutations identified may be neutral, but some may affect the outcome of subsequent experiments performed to analyze the phenotypes. Our findings indicate that all generated mutations should undergo complete genomic sequencing. The information gained may deepen our understanding of bacterial life processes.
The global transcription regulator CRP protein controls diverse cellular processes in many bacteria. Our group previously reported that Tn4531 insertion in FIP52_09345 (designated as crpR1), which encodes a special CRP protein without the HTH motif, attenuated the virulence of Riemerella anatipestifer. Here, sequence analyses indicate that R. anatipestifer crpR1 can be classified into three subtypes (crpR1a, crpR1b, and crpR1c) based on the ORF length. Of 52 strains, 42 carried crpR1a. CrpR1b and CrpR1c contained both the CRP domain and HTH motif, while CrpR1a lacked the HTH motif. The crpR1a sequence had two alleles, and crpR1b of some strains originated from allele 1 of crpR1a, while crpR1c of strain CH3 originated from allele 2 of crpR1a. Point mutations and insertion sequences may play an important role in the evolution of crpR1. Moreover, deletion of crpR1a from strain Yb2 resulted in reduced virulence in ducklings by approximately 1,000-fold, while deletion of the 3'-end of crpR1a (encoding the disordered region) or the downstream AS87_RS10485 (encoding the HTH motif) resulted in reduced virulence of strain Yb2 in ducklings. In addition, mutation of 450C451A in the crpR1a ORF of strain Yb2 to 450G generated the non-pathogenic Yb2 crpR1-450G mutant, with the crpR1 subtype changed to crpR1b. Mutants with the 3' end of the crpR1 ORF in Yb2 crpR1-450G, consistent with that of strain HXb2 or CH3 crpR1, were pathogenic to ducklings. These findings confirm that adaptive evolution resulted in three subtypes of R. anatipestifer crpR1.IMPORTANCEBacterial CRP regulators regulate metabolism, stress resistance, biofilm formation, and pathogenesis. Here, we report that a special CRP in Riemerella anatipestifer, CrpR1a, which lacks the HTH motif, has biological functions. The crpR1a, carried by most R. anatipestifer strains, is the ancestor of crpR1b and crpR1c. Deletion of crpR1a in strain Yb2 or mutation of 450C451A to 450G in this crpR1a ORF leads to significant reduction or loss of virulence, but a single base mutation of 559C > A, 586C > T, or 591T > C in the non-pathogenic mutant Yb2 crpR1-450G crpR1 ORF increases its virulence in ducklings. Moreover, deletion of the C-terminal disordered region of CrpR1a or its downstream HTH motif resulted in reduced virulence of strain Yb2.
Health monitoring based on post-mortem examination is essential for the management of endangered animal species. This is especially true for reintroduced species living in small populations with low genetic diversity, such as the Eurasian lynx (Lynx lynx) in Switzerland. Thanks to systematic post-mortem examinations, the Institute for Fish and Wildlife Health (FIWI), University of Bern, has acquired a comprehensive view of the lynx health in Switzerland. This study provides an updated overview of the causes of morbidity and mortality in the Eurasian lynx in Switzerland from 2000 to 2022. A total of 346 necropsied lynx (found dead, euthanized, or culled) were included in this study, and a cause of death (COD) was identified in 318 of them (91.9%). Overall, the main COD was blunt trauma (n = 183, 52.9% - largely vehicular collision). Starvation, resulting from the separation of dependent juveniles from their mother, was the second most frequent COD (n = 63, 18.2%). Fatal infectious diseases were relatively low (n = 32, 9.2%). However, we documented some significant pathogens such as canine distemper virus (CDV) and metastrongyloid nematodes. Illegal killing was confirmed in 23 cases (6.6%). Of note, illegal killing is likely underestimated in this study, given that radio-collared lynx were found to be proportionally more often illegally killed than the unmonitored ones found by chance. Furthermore, most individuals were found to be affected at least by one non-specific, mild to moderate inflammatory process of unknown origin, such as interstitial pneumonia (n = 59) or interstitial nephritis (n = 25). Additionally, cardiac changes of variable severity were observed in 125 lynx, and severe soft tissue mineralization was detected in 10 individuals. The frequency of these findings warrants further investigation. Thus, this study confirms the importance of systemic post-mortem examination and general health surveillance of free-ranging Eurasian lynx in Switzerland, in support of translocation projects, conservation of the species, and to provide a better understanding of their pathologies.
There is a growing understanding that slow growth and dormancy due to nutrient deprivation are very common physiological states exhibited by bacterial communities in a myriad of environments. However, very little is known about the role of slow growth and dormancy in biofilm regulation. Here, we utilize tractable dormancy and aggregation assays in nontuberculous mycobacteria (NTM) to ask the fundamental question of how growth arrest impacts the processes of aggregation and dispersal. First, we show that the well-conserved dormancy regulator DosSR affects biofilm formation in Mycobacterium smegmatis, as a dosR deletion mutant undergoes spurious re-aggregation and dispersal during aerobic late stationary phase. Identification of a suppressor mutation blocking re-aggregation in the ΔdosR mutant allowed us to determine a role for the antibiotic resistance factor WhiB7 in driving re-aggregation in M. smegmatis. We utilized BioOrthogonal NonCanonical Amino acid Tagging (BONCAT), qPCR, and quantitative aggregation assays to build a model wherein reductive stress in ΔdosR potentiates stationary phase translation in a WhiB7-dependent manner, permitting aggregation in dormant stationary phase cells. In addition, during stationary phase, WhiB7-activating reducing agents and antibiotics could trigger re-aggregation in both wild-type M. smegmatis and clinical isolates of the opportunistic NTM pathogen Mycobacterium abscessus. Finally, we determined that, in contrast to aerobic stationary phase, M. smegmatis does not aggregate or disperse in response to chemical cues or antibiotics under the Wayne model of hypoxic dormancy. Our work reveals a regulatory interaction between dormancy and aggregation that could have broad implications for treating and preventing NTM biofilms.IMPORTANCEMycobacteria aggregate to form multicellular biofilms that provide protection from external stressors and increase antibiotic tolerance. Understanding the pathways regulating biofilm formation can aid the identification of useful targets for developing new drugs. With a growing appreciation that pathogens are often in a slow growth/dormant state during infection, we investigate how dormancy affects biofilm formation and dispersal in two nontuberculous mycobacteria (NTM) species: Mycobacterium smegmatis and the opportunistic pathogen Mycobacterium abscessus. We find that activation of the WhiB7-mediated antibiotic response permits biofilm formation in aerobic stationary phase by reinitiating protein synthesis; however, cells under hypoxic dormancy are unresponsive. Our work provides important context to combatting biofilm formation in infection sites, informing future studies and aiding design of biofilm dispersal agents.
Bacterial infections often occur in polymicrobial biofilms where nutrient limitation and interspecies interactions can profoundly shape microbial physiology. Enterococcus faecalis can antagonize Pseudomonas aeruginosa growth under conditions of iron limitation, such as those found in the mammalian host. Here, we report that this growth antagonism reveals surviving P. aeruginosa cells capable of surviving antibiotic challenge, including ampicillin, cefepime, and ciprofloxacin, when grown in iron-restricted biofilms with E. faecalis. Transcriptomic profiling of P. aeruginosa revealed a distinctive response characterized by broad downregulation of biosynthetic, metabolic, and virulence pathways, alongside selective induction of membrane remodeling proteins, transport systems, and biofilm-associated genes. Induction of arnT in P. aeruginosa, required for lipid A modification, correlated with enhanced antibiotic survival to ampicillin, cefepime, and ciprofloxacin. Additionally, the diguanylate cyclase SiaD and efflux transporter MfsC in P. aeruginosa were implicated in decreased antibiotic susceptibility to the same antibiotics. This transcriptional response was unique to the dual stress of iron deprivation and microbial competition with E. faecalis, illustrating how interspecies interactions can simultaneously inhibit and protect P. aeruginosa, shedding light on potential persistence mechanisms in iron-limited polymicrobial environments.IMPORTANCEThis study addresses antibiotic susceptibility in Pseudomonas aeruginosa, a major opportunistic ESKAPE pathogen, within polymicrobial biofilms and under host-relevant iron-restricted conditions. Polymicrobial biofilm-associated infections are notoriously difficult to treat due to complex interspecies interactions and increased antibiotic resilience. We demonstrate that Enterococcus faecalis not only antagonizes P. aeruginosa growth under iron limitation but also induces a unique transcriptional profile, enhancing P. aeruginosa survival during antibiotic challenge. This shift involves broad transcriptional reprogramming in P. aeruginosa, characterized by global metabolic downregulation and activation of envelope remodeling pathways, including the arn operon. These findings reveal how interspecies interactions under iron stress can both suppress and protect bacterial pathogens and underscore the importance of considering community context in treatment strategies for persistent infections.
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that requires iron to cause infection. Iron can also be toxic due to its participation in Fenton chemistry, resulting in the production of reactive oxygen species (ROS). Thus, P. aeruginosa regulates the uptake, use, and storage of iron to mitigate the effects of ROS. P. aeruginosa uses several mechanisms to manage oxidative stress, including superoxide dismutases, catalases, and members of the ferritin superfamily. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are predicted to pair with and destabilize mRNA transcripts for several oxidative stress response proteins, including sodB, katA, and brnD, encoding a novel bacterioferritin-like Dps protein. In this study, we developed brnD reporter constructs that are responsive to PrrF-mediated iron regulation. We demonstrated that PrrF-mediated regulation of brnD occurs in the 5' untranslated region of its mRNA, likely via a conserved region of complementarity with the PrrF sRNAs. We further showed that brnD mRNA levels are increased upon hydrogen peroxide treatment. Surprisingly, peroxide treatment also resulted in elevated levels of the PrrF sRNAs. Last, we investigated these regulatory effects in Pseudomonas fluorescens, revealing similar iron regulation of the PrrF sRNAs and brnD ortholog, as well as peroxide-induced expression of the PrrF sRNAs. Combined, these data highlight a distinct class of iron-responsive ferritin family proteins with potential functional conservation across the pseudomonads, and they reveal a novel aspect of the oxidative stress response involving the PrrF sRNAs. Iron is a crucial micronutrient for Pseudomonas aeruginosa survival and virulence, yet it can also be toxic due to the production of reactive oxygen species. The PrrF small regulatory RNAs (sRNAs) are transcribed in iron-limiting conditions and block the expression of several mRNAs involved in P. aeruginosa's oxidative stress response. Here, we make the surprising discovery that oxidative stress induces expression of the PrrF sRNAs. We also provide evidence that PrrF regulation of two mRNA targets is hindered upon oxidative stress. Oxidative stress similarly induces the PrrF sRNAs in Pseudomonas fluorescens, indicating conservation of this novel regulatory event. This study, therefore, highlights a novel and conserved regulatory link between iron homeostasis and oxidative stress protection in the pseudomonads.
Thermoanaerobacterium saccharolyticum is an anaerobic, thermophilic bacterium that has been proposed for use in consolidated bioprocessing in coculture with cellulolytic bacteria such as Clostridium thermocellum for ethanol production. Although the mixed acid fermentation of both species has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far by C. thermocellum is half that produced by T. saccharolyticum. This has motivated us to understand the mechanistic basis of the robust T. saccharolyticum ethanol pathway, so that key features can be recapitulated in C. thermocellum. Previously, we characterized the individual role of the main genes responsible for electron transfer in the ethanol production of T. saccharolyticum. However, the consequences of the combined loss of function of these genes has not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. Here, we combined knockouts of ferredoxin nicotinamide oxidoreductases (nfnA and nfnB) and hydrogenases (hydA and hfsD). We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth and observed a mutation in the alcohol dehydrogenase adhA gene. We determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of electron transfer pathways in this organism.IMPORTANCEThermoanaerobacterium saccharolyticum has the potential to be used for the conversion of lignocellulose-derived sugars into bioethanol via consolidated bioprocessing in cocultures with Clostridium thermocellum or via transfer of its ethanol production pathway to the same bacterium. However, attempts to transfer the pathway to increase ethanol titer have not yet been successful. A deeper understanding of how electron transfer pathways operate in this thermophile and how they adapt to metabolic disturbances (such as the absence of metabolic pathways for ferredoxin oxidation, for example) will improve our ability to engineer this organism for increased product formation and improve our ability to transfer its remarkable ethanol production phenotype to other microbes. These, in turn, have potential benefits for sustainable production of fuels and chemicals.
Streptococcus mutans is a primary architect of dental caries, utilizing complex genetic networks to build resilient, acid-producing biofilms. While pooled screens (Tn-seq) have identified important fitness factors, they often fail to capture extracellular or moderate-effect determinants due to community-level masking. Therefore, to study biofilm phenotypes, we constructed a comprehensive arrayed library of 9,216 mutants and used Cartesian Pooling-Coordinate Sequencing (CP-CSeq) to establish a sequence-defined resource covering 51% of non-essential genes. By screening the entire collection in isolation, we identified several novel biofilm determinants, including the putative metal transporter SMU_635 and the glycosylation-associated protein SMU_2160. However, systematic whole-genome sequencing (WGS) of our hits revealed an interesting level of genomic instability: 25% of biofilm-defective mutants had undergone spontaneous recombination at the gtfBC locus, while 7% had lost the TnSmu1 element, an excision rate 1,000-fold higher than previously reported. While targeted mutagenesis confirmed that TnSmu1 loss does not impact biofilm integrity, the gtfBC deletions directly accounted for the most severe phenotypes, highlighting a systemic risk of misattributing gene functions to primary transposon insertions. Our findings provide a powerful new genetic resource for the S. mutans community while establishing a critical new standard: an arrayed library is only as defined as its underlying genome, making systematic genomic verification an essential prerequisite for accurate functional genomics. Streptococcus mutans causes dental caries through resilient, acidogenic biofilm formation. While pooled screens often overlook extracellular or moderate-effect determinants due to community masking, this study presents a sequence-defined arrayed mutant library to dissect individual gene functions in isolation. Beyond known machinery, we identified novel biofilm determinants, including metal transporter SMU_635 and glycosylation-associated protein SMU_2160. Crucially, we uncovered pervasive genomic instability at the gtfBC and TnSmu1 loci. This reveals a systemic functional genomics risk: misattributing phenotypes to primary mutations when backgrounds undergo large-scale rearrangements. By establishing whole-genome verification as a necessary standard, this research ensures that future therapeutic target identification is built upon a verified genetic foundation.
Vibrio parahaemolyticus, a gram-negative marine bacterium, is a major cause of seafood-borne gastroenteritis worldwide. This pathogen relies on type III secretion system 2 (T3SS2), which is encoded on a pathogenicity island, for its enteropathogenicity. Expression of T3SS2 is activated by a regulatory pathway centered on the transcriptional activator VtrB, which is antagonized by the xenogeneic silencer, histone-like nucleoid-structuring protein (H-NS). However, the complete transcriptional network is not yet fully understood. In this study, we identified TsrA as a negative regulator of T3SS2 gene expression. TsrA is a small protein conserved among Vibrio species that lacks a putative DNA-binding motif but has been implicated in the regulation of virulence genes in Vibrio cholerae. In V. parahaemolyticus, deletion of tsrA increased VtrB production and T3SS2 secretion, thereby enhancing T3SS2-dependent pathogenicity. Transcription of vtrB occurs via a two-step activation process, in which TsrA affects the primary activation step, thereby modulating VtrB production. We further provide experimental evidence that TsrA physically interacts with H-NS via its C-terminal region, which correlates with its regulatory activity on vtrB expression. A systematic mutational analysis of the C-terminal 26 residues revealed several residues critical for TsrA regulatory activity. Moreover, the regulatory effect of TsrA on T3SS2 gene expression was dependent on H-NS, demonstrating that TsrA functions in concert with H-NS. Thus, our findings provide new insights into the regulatory mechanisms of virulence gene expression in V. parahaemolyticus by defining the role of TsrA in this network, while also placing TsrA among H-NS co-regulators.IMPORTANCENucleoid-associated proteins (NAPs) play key roles in virulence gene regulation in bacteria. The best-studied NAP is H-NS, which often functions with co-regulators to fine-tune gene expression. TsrA, a small protein lacking a DNA-binding motif conserved among Vibrio species, has been suggested to be functionally related to H-NS in Vibrio cholerae, although its mechanism remains unknown. Here, we demonstrate that TsrA negatively regulates the expression of type III secretion system 2 (T3SS2), a major virulence determinant of Vibrio parahaemolyticus, an important seafood-borne pathogen. TsrA modulates the transcription of vtrB, which encodes the essential activator for T3SS2 expression, through direct physical interaction with H-NS. Our findings reveal a molecular link between TsrA and H-NS, providing mechanistic insights into NAP- and TsrA-mediated regulation of virulence in Vibrio.
Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, uses a cell-cell signaling system called quorum sensing to coordinate group behaviors. Quorum sensing in P. aeruginosa is a model to study cooperative behaviors in populations. Several studies of cooperation have been conducted using strain PAO1. Wild-type PAO1 harbors a mutation in mexS, which encodes a negative regulator of the transcription factor MexT, which, in turn, activates many genes, including the efflux pump MexEF-OprN. We hypothesized that the PAO1 mexS mutation might affect cooperative behaviors. When P. aeruginosa is passaged daily on casein as a sole carbon source, quorum sensing is required to induce synthesis of the extracellular proteases needed to acquire carbon and energy. When PAO1 is grown on casein, individuals with inactivating mutations in the quorum-sensing regulator LasR are reproducibly enriched in the population. These LasR mutants are cheaters that benefit from cooperatively produced proteases and have a fitness advantage over cooperators. We passaged wild-type PAO1, PAO1 with a gene-corrected version of mexS, or PAO1 with a null mutant of mexT on casein as the sole carbon and energy source. We found that correcting the mexS mutation resulted in unstable cooperation: bacterial cultures failed to propagate after about 15 days, whereas the wild type propagated for the duration of our 30-day experiment. The MexS-corrected and MexT-deficient populations also reproducibly exhibited emergence of a particular quorum-sensing variant, LasR-V226I. This variant activated a subset of quorum-sensing regulated genes, suggesting an evolutionary pathway to alter P. aeruginosa quorum-sensing regulons.IMPORTANCEQuorum sensing governs cooperative activities and has been used as a model for studying cooperative behaviors in bacterial populations. Pseudomonas aeruginosa has two interlinked acyl-homoserine lactone quorum-sensing circuits, LasR-I and RhlR-I; the relationship between these circuits is influenced by the regulator MexT. In the well-studied strain PAO1, inactivation of MexT results in enhanced activity of the quorum-sensing transcription factor, RhlR, and we found that it also destabilized cooperative behaviors in this bacterium. MexT-inactive strains also reproducibly support the emergence of a specific LasR variant, V226I, of P. aeruginosa, offering insight into evolutionary pressures that select for mutations in quorum-sensing transcription factors and, by extension, a method for bacteria to alter the cohort of genes that are quorum-controlled.
tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Several studies have reported that tusA deficiency affects iron-sulfur (Fe-S) homeostasis in addition to tRNA sulfur modification, resulting in pleiotropic phenotypes. In this study, we analyzed the phenotype of tusA-deficient Escherichia coli and its underlying mechanisms. Although the Keio tusA knockout strain showed increased swimming motility and flagellar biosynthesis, these phenotypes were not restored by tusA complementation. Genome resequencing of the original Keio tusA knockout strain identified an unintended secondary mutation in lrhA, a transcriptional regulator of flagellar and chemotaxis genes. This secondary mutation impaired lrhA function, contributing to enhanced flagellar synthesis and swimming motility, as well as altered global gene expression. A tusA knockout strain in which the secondary mutation was corrected (ΔtusA) exhibited reduced swimming motility compared with the wild-type strain, despite showing no abnormalities in flagellar formation. Furthermore, ΔtusA displayed increased resistance to cationic antibacterial agents, including cetyltrimethylammonium bromide, cetylpyridinium chloride, and protamine sulfate. The reduced motility caused by tusA deletion was observed independently of Fur, a global regulator of iron homeostasis, whereas resistance to cationic antibacterial agents was abolished in the fur knockout background. In addition, altered expression of outer membrane protein (omp) genes was observed in ΔtusA, and deletion of these omp genes abolished resistance to cationic antibacterial agents. Together, these results indicate that, by disentangling the phenotypic effects of tusA deletion from those of the unintended secondary mutation, loss of tusA confers resistance to cationic antibacterial agents through a Fur-dependent and Omp-dependent mechanism.IMPORTANCEtRNA 2-thiouridine synthesizing protein A (TusA) is a sulfur-carrier protein involved in tRNA thiolation and iron-sulfur (Fe-S) cluster homeostasis. Accordingly, deletion of tusA results in pleiotropic phenotypes. The Keio tusA knockout strain is widely used to study tusA function; however, resequencing revealed a secondary mutation in lrhA, a transcriptional regulator of flagellar biosynthesis and chemotaxis. This lrhA mutation affected flagellar formation, swimming motility, and gene expression in the Keio tusA knockout strain. We further demonstrated that a tusA knockout strain with repaired lrhA exhibited reduced swimming motility and increased resistance to cationic antimicrobial agents. Our findings highlight the impact of the secondary mutation in the widely used Keio tusA knockout strain and provide insights into the functions of tusA.
Iron availability affects oceanic primary productivity and the strength of the biological carbon pump. However, the mechanism of iron solubility in hypersaline environments is poorly understood. In this work, we investigated the extremely halophilic archaeon Haloferax volcanii to reduce ferrihydrite under 25% salinity, aiming to elucidate its energy metabolism associated with extracellular electron transfer (EET). Ferrihydrite reduction by H. volcanii reached a plateau around the eighth day, with an Fe(III) conversion of approximately 41%. The redox-active substances on the cell surface play a central role via EET. Proteomic analysis revealed that the relative abundance of membrane-bound quinoline b-type cytochrome oxidases (A0A841HB82, A0A841HBL7) increased by 73.8% and 120%, respectively, when H. volcanii used ferrihydrite rather than O2 as the terminal electron acceptor. Moreover, H. volcanii was found to secrete riboflavin, which can function as an electron shuttle mediating indirect EET. Our findings provide insights into the survival and evolutionary strategies of early life in anaerobic environments on Earth, and the EET capability of H. volcanii highlights its potential applicability in the treatment of pollutants in hypersaline wastewater.IMPORTANCEAs global warming intensifies, increased evaporation and seawater intrusion are leading to the expansion of high-salinity environments. Dissolved iron in these environments plays a crucial role in regulating the biogeochemical carbon cycle, thereby influencing global carbon dynamics. Microbial dissimilatory Fe(III) reduction may be the primary process driving the biogeochemical cycling of iron in such hypersaline environments. This study demonstrates that the extreme halophile Haloferax volcanii reduces ferrihydrite under high-salinity, anaerobic conditions. This reduction occurs primarily via membrane-associated cytochromes and secreted riboflavin. Consequently, investigating the extracellular electron transfer (EET) capabilities of halophilic archaea may provide new insights into the origin and maintenance of bioavailable iron under high-salinity stress.
Bronchiectasis is a progressive pulmonary disease with repeated cough, expectoration and frequent respiratory infections. Every patient should have sample collected for routine bacteriological culture. Determining the disease's severity can help with therapy and follow-up choices. To detect the bacteriology of bronchiectasis patients and relation to disease severity. 60 patients with bronchiectasis exacerbation were investigated at chest department of Fayoum University Hospital. Broncho alveolar lavage for culture and sensitivity was done. Disease severity was assessed by cough score, mMRC dyspnea score, oxygen saturation, no of lobes affected in CT chest and modified rieff score, Spirometry and classification of severity by FEV1, and finally FACED and BSI scores were calculated. Isolation of H.Influenza represents 40%, Pseudomonas represents 26.7%, Klebsiella represents 20%, Staph aureus represents 10% and Pseudomonas& Klebsiella represent 3.3%. There was a statistically significant lower mean of oxygen saturation in cases infected with both pseudomonas and Klebsiella. There was a statistically significant high percentage of mild Modified Reiff score among cases infected with H. influenza, moderate degree among cases infected with Klebsiella, but severe degree among cases infected with pseudomonas. H. influenzae consider as a major pathogen isolated by BAL culture in patients with bronchiectasis exacerbation, followed by P. aeruginosa, then klebsiella then S. aureus. Cases infected with P. aeruginosa and klebsiella have the worst oxygen saturation. The highest modified Rieff score was in P. aeruginosa than other isolated organisms.
The genus Akkermansia was first described in 2004 following the identification of Akkermansia muciniphila, a Gram-negative, mucin-degrading bacterium of the intestine that constitutes 1-3% of the total adult fecal content. Since the interest in A. muciniphila in human health has increased over the past decade, an extensive amount of research examining the impact of A. muciniphila on metabolic disorders, non-communicable diseases, and during infection has been published. Furthermore, a rapidly evolving area of research is the role of A. muciniphila in gynecological health. Many studies have shown that the presence of A. muciniphila may decrease the chances of negative health outcomes. Some of these protective effects include enhancement of epithelial barrier integrity and metabolism, immune modulation, and attenuation of inflammatory responses. As such, A. muciniphila has gained significant interest for its promising role as a next-generation probiotic. Notably, most of the in vivo evidence reviewed here demonstrates the probiotic potential of A. muciniphila. However, some findings suggest that its role is context-dependent, which may be influenced by the type of infection, diet, and microbiota composition. Herein, we review associations between Akkermansia species and an array of infectious diseases caused by diverse pathogen classes, including bacteria, viruses, fungi, and parasites. We also review the impact of Akkermansia species in gynecological conditions, particularly during pregnancy. The emerging role of A. muciniphila in promoting health, and in some cases disease, has important implications for understanding complex microbial-host interactions, as well as for the development of novel therapeutics.
Bacillus subtilis is a model for cell differentiation, capable of transitioning between distinct states: a sessile, chained state (often associated with biofilm formation), and a motile, planktonic state. This transition is governed by a complex regulatory network that includes alternative sigma factor-D (σD), which drives the expression of genes for autolysins, flagellar biosynthesis, and chemotaxis, resulting in separated, flagellated, motile cells. Although the conserved transcription-coupled repair factor mutation frequency decline (Mfd) is best known for resolving stalled RNA polymerase (RNAP) during DNA repair, its involvement in cell differentiation during the stationary phase remains poorly understood. This research shows that in the absence of Mfd translocation or RNAP recruitment, RNAP completes transcription of motility genes more frequently, despite unchanged σD transcript levels. Increased transcription of the σD-dependent motility regulon led to greater flagellation; however, swimming motility and pH taxis were paradoxically reduced, indicating that Mfd is required to translate flagellar gene expression into functional motility. Notably, Mfd-deficient cells failed to maintain chained subpopulations, indicating an additional role in sustaining population heterogeneity. These findings reveal a previously unrecognized function of Mfd as an RNAP-modulating factor that coordinates motility gene expression, thereby expanding our understanding of how transcription-coupled repair proteins influence bacterial physiology and behavior under non-proliferative conditions.IMPORTANCEThe mutation frequency decline (Mfd) enzyme mediates transcription-coupled repair in transcribed genes by directly interacting with RNA polymerase (RNAP) during transcription of coding sequences. However, whether the Mfd factor regulates gene expression associated with adaptations unrelated to DNA repair and mutagenesis remains vague. Here, we show that Mfd regulates the completion of full transcripts of genes that confer swimming motility, chemotaxis, and cell heterogeneity in Bacillus subtilis. Furthermore, we identified the Mfd's translocase activity and its interaction with RNAP as key elements of this regulation. Therefore, Mfd's importance in bacterial physiology and adaptation goes beyond DNA repair.
Contractile injection systems (CISs) are derivatives of phage tails and are widely distributed in prokaryotes. CISs load cognate effectors and eject them through contractile actions resembling those of phage tails. Ejected effectors play central roles in CIS functionality by acting on target cells and mediating various biological processes. Here, we report a novel group of CIS effectors related to phage tape measure protein, the transmembrane component of the phage infection machinery. This group is broadly distributed within the class Actinobacteria, one of the bacterial classes in which CIS gene clusters are highly conserved, and is represented by Sle1, a cognate effector of the intracellularly localized Streptomyces lividans phage tail-like nanoparticle (SLP). This effector is associated with Sle2, which contains a CIS effector core domain and interacts with the SLP core component. Sle1 is packaged inside SLP and is translocated to lipid membranes along with SLPs. The functional domain of Sle1 enriches the membrane-associated subproteome in S. lividans and E. coli. This effect modifies the physiological properties of S. lividans, ultimately enhancing its adaptation to microbial competition. In addition, we revealed that Sle1-type effectors conserved among actinobacterial species are structurally and functionally diverse in their functional domains. One of them from Micromonospora eburnea constitutes a novel toxin-antitoxin system, and introducing its functional domain into Sle1 reprograms the phenotypic responsiveness of S. lividans to neighboring bacteria. Our findings illustrate that phage elements can be incorporated into CISs as reconfigurable platforms for bacterial adaptation to various environmental conditions.IMPORTANCEBacterial CISs have attracted interest for their importance in microbial ecology and potential in biotechnological applications. However, understanding of their functional diversity is currently limited because many CIS effectors remain unannotated due to a lack of inferable structural and genetic signatures. Our findings on Sle1 and its relatives illuminate a previously unidentified class of CIS effectors with phage tape measure protein-related modular architecture, association with the CIS effector core domain, and wide distribution within the major class of Actinobacteria, substantially expanding the known repertoire of effector classes. The impact of Sle1 on S. lividans suggests a link between CIS effectors and bacterial adaptation to environmental conditions, highlighting unexplored functional diversity of CIS effectors as tuners of bacterial phenotypes in communities.
Cyclic di-GMP (c-di-GMP) is a key bacterial second messenger that regulates a wide range of cellular processes, including biofilm formation and virulence. Multi-domain one-component systems regulate c-di-GMP synthesis and turnover in response to external signals. Periplasmic sensing of environmental cues is performed by versatile but conserved sensory domains, including members of the CHASE4 superfamily. Here, we explore the promiscuity of the CHASE4 domain in c-di-GMP signal transduction in Pseudomonas aeruginosa by analyzing two CHASE4-containing transducers from the virulent PA14 strain, namely PA14_53310 and PA14_37690. With the integration of biochemical and biophysical methods, such as UV-Vis spectroscopy, circular dichroism, and isothermal titration calorimetry, we demonstrate that the CHASE4 domains of these proteins possess different ligand specificities. We show that PA14_53310 is a diguanylate cyclase, and that its enzyme activity is controlled by heme binding to its periplasmic CHASE4 domain. The PA14_37690 CHASE4 domain, on the other hand, does not bind heme, but likely recognizes copper, indicating a role in metal ion sensing. A comparison with the PAO1 counterpart, that is, PA0847 and PA2072, is discussed and the divergences highlighted. This work demonstrates that the CHASE4 domain is a multifunctional sensory module that can be calibrated to detect a range of environmental cues, thus providing a mechanism for c-di-GMP signaling refinement in P. aeruginosa. These results shed light on the molecular basis for the functional diversification of CHASE4-containing one-component systems and their role in bacterial adaptation.IMPORTANCEThe results presented in this study reveal a fascinating versatility of the CHASE4 domain in PA14 Pseudomonas aeruginosa cyclic di-GMP signaling systems (i.e., PA14_53310 and PA14_37690), highlighting its ability to sense different environmental cues, such as heme and copper. In the context of infection, the interplay between heme and copper sensing could be particularly relevant, being both nutrients or poisons. Their sensing (and adaptive response) would be a significant advantage for the bacterium, allowing it to optimize its virulence and survival strategies. Therefore, these results represent the starting point for future in vivo studies aimed at exploring the relevance of heme and copper in pathogenesis, to finally develop novel anti-virulence strategies targeting the pathogen's ability to adapt to the host environment.
Formate hydrogenlyase (FHL) enzymes are evolutionarily related to mitochondrial complex I. Formate-inducible FHL-1 of Escherichia coli catalyzes disproportionation of formic acid into H2 and CO2 during mixed-acid fermentation. In contrast to FHL-2 enzymes found in many microorganisms, including E. coli, FHL-1 shares only two of the usual five membrane-domain subunits, perhaps explaining its apparent inability to translocate protons. This raises the question, however, as to the physiological function of the FHL-1 enzyme. Here, we demonstrate that FHL-1 serves to regulate intracellular pH and CO2 levels in stationary-phase fermenting cells. Determination of intracellular and extracellular pH values in E. coli strains with and without an FHL-1 enzyme revealed an alkalinization (0.5 pH units) of the cytoplasm in cells lacking FHL-1. This increase in pHi correlated with a concomitant threefold decrease in the ATP concentration in these cells, while the parental strain retained both high ATP levels and a pHi of 6.7. The reduction in ATP levels in the mutant correlated with an increase in F1Fo-ATPase activity. Remarkably, ATP levels and pHi in the FHL-1-negative mutant could be restored to near wild-type levels by growth in the presence of bicarbonate. Together, these data indicate that FHL-1 functions to provide CO2/bicarbonate for key carboxylation reactions, concomitantly maintaining intracellular pH homeostasis and thereby indirectly optimizing ATP levels by preventing excessive ATP hydrolysis via F1Fo-ATPase in its attempt to neutralize pHi. CO2 production by cytoplasmically oriented FHL-1 substitutes for a pyruvate decarboxylation reaction, and explains why maintaining formate homeostasis is central to mixed-acid fermentation. The signature molecule of enterobacterial mixed-acid fermentation, formate, is disproportionated into H2 and CO2 by formate hydrogenlyase (FHL), an evolutionarily ancient enzyme phylogenetically related to proton-translocating complex I. However, as FHL-1 of Escherichia coli lacks the capacity to translocate protons, its physiological role remains unclear. We show that FHL-1 functions to maintain pH homeostasis and intracellular CO2/bicarbonate levels in stationary-phase cells. Mutants lacking a functional FHL-1 fail to generate H2 and CO2, causing activation of F1Fo-ATPase, which hydrolyzes ATP to maintain neutral cytoplasmic pH. The balance between formate synthesis and its intracellular disproportionation is necessary to maintain the supply of bicarbonate for carboxylation reactions, thus ensuring pH homeostasis and ultimately optimal ATP levels, which together aid stationary-phase survival.
Uropathogenic Escherichia coli (UPEC) is the leading causative agent of urinary tract infection (UTI). Copper (Cu) is a host effector mobilized to the bladder during the innate immune response activated by acute UTI. E. coli precisely regulates Cu homeostasis via efflux systems because excess Cu is toxic. Long-chain fatty acid (LCFA) homeostasis is critical for the survival of E. coli during envelope stress. However, the interaction between LCFA metabolism and survival during Cu stress, which is also known to induce envelope stress, is not known and is the subject of this report. We hypothesized that fatty acid homeostasis is critical for the survival of UPEC during Cu toxicity and modulates UPEC virulence during UTI. A reverse genetic screen of the KEIO collection identified FabR and FadR as critical proteins for optimal survival of E. coli during Cu stress. FadR and FabR regulate transcription genes involved in LCFA metabolism. By using targeted deletion mutants and genetic complementation, we demonstrate that LCFA metabolism affects survival of UPEC during Cu stress. Cell-associated Cu concentrations were decreased in the ΔfadR mutant, and we found that the expression of Cu detoxification genes was upregulated in these mutants. UPEC lacking FadR were more sensitive to superoxide stress compared to the wild-type strain. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that lipid saturation levels were increased in UPEC during Cu stress in a fabR- and fadR-dependent manner. Furthermore, both mutants displayed lower flagellar expression and attenuated fitness during UTI in the mouse model. Together, our findings demonstrate that LCFA homeostasis is a crucial mediator of Cu homeostasis and UPEC virulence during UTI.IMPORTANCEUropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infection (UTI). Previous research has established that UPEC experiences copper stress during UTI, and resistance to copper is critical for UPEC virulence. Envelope stress response systems are activated by copper (Cu) stress, and long-chain fatty acid (LCFA) homeostasis is critical for the survival of E. coli during envelope stress. However, the interaction between LCFA metabolism and Cu stress is not known. Our results demonstrate that LCFA homeostasis is a crucial mediator of copper homeostasis in UPEC and modulates UPEC virulence during UTI.