Deletion Of Operon Research Articles

The Pseudomonas aeruginosa sphBC genes are important for growth in the presence of sphingosine by promoting sphingosine metabolism.

Sphingoid bases, including sphingosine, are important components of the antimicrobial barrier at epithelial surfaces where they can cause growth inhibition and killing of susceptible bacteria. Pseudomonas aeruginosa is a common opportunistic pathogen that is less susceptible to sphingosine than many Gram-negative bacteria. Here, we determined that deletion of the sphBCD operon reduced growth in the presence of sphingosine. Using deletion mutants, complementation, and growth assays in P. aeruginosa PAO1, we determined that the sphC and sphB genes, encoding a periplasmic oxidase and periplasmic cytochrome c, respectively, were important for growth on sphingosine, while sphD was dispensable under these conditions. Deletion of sphBCD in P. aeruginosa PA14, P. protegens Pf-5, and P. fluorescens Pf01 also showed reduced growth in the presence of sphingosine. The P. aeruginosa sphBC genes were also important for growth in the presence of two other sphingoid bases, phytosphingosine and sphinganine. In wild-type P. aeruginosa, sphingosine is metabolized to an unknown non-inhibitory product, as sphingosine concentrations drop in the culture. However, in the absence of sphBC, sphingosine accumulates, pointing to SphC and SphB as having a role in sphingosine metabolism. Finally, metabolism of sphingosine by wild-type P. aeruginosa protected susceptible cells from full growth inhibition by sphingosine, pointing to a role for sphingosine metabolism as a public good. This work shows that metabolism of sphingosine by P. aeruginosa presents a novel pathway by which bacteria can alter host-derived sphingolipids, but it remains an open question whether SphB and SphC act directly on sphingosine.

The Bacillus subtilis yqgC-sodA operon protects magnesium-dependent enzymes by supporting manganese efflux.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function, and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations, they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the YS deletion mutants results from distinct enzymatic vulnerabilities.IMPORTANCEBacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. In Bacillus subtilis, Mn-dependent superoxide dismutase (MnSOD) is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual proteins) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Nutrient Availability and Biofilm Polysaccharide Shape the Bacillaene-Dependent Antagonism of Bacillus subtilis against Salmonella Typhimurium.

Salmonella enterica is one of the most common foodborne pathogens and, due to the spread of antibiotic resistance, new antimicrobial strategies are urgently needed to control it. In this study, we explored the probiotic potential of Bacillus subtilis PS-216 and elucidated the mechanisms that underlie the interactions between this soil isolate and the model pathogenic strain S. Typhimurium SL1344. The results reveal that B. subtilis PS-216 inhibits the growth and biofilm formation of S. Typhimurium through the production of the pks cluster-dependent polyketide bacillaene. The presence of S. Typhimurium enhanced the activity of the PpksC promoter that controls bacillaene production, suggesting that B. subtilis senses and responds to Salmonella. The level of Salmonella inhibition, overall PpksC activity, and PpksC induction by Salmonella were all higher in nutrient-rich conditions than in nutrient-depleted conditions. Although eliminating the extracellular polysaccharide production of B. subtilis via deletion of the epsA-O operon had no significant effect on inhibitory activity against Salmonella in nutrient-rich conditions, this deletion mutant showed an enhanced antagonism against Salmonella in nutrient-depleted conditions, revealing an intricate relationship between exopolysaccharide production, nutrient availability, and bacillaene synthesis. Overall, this work provides evidence on the regulatory role of nutrient availability, sensing of the competitor, and EpsA-O polysaccharide in the social outcome of bacillaene-dependent competition between B. subtilis and S. Typhimurium. IMPORTANCE Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica, which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors. Moreover, the production of extracellular polysaccharides by B. subtilis further alters the influence of these factors. Hence, this work highlights the inhibitory effect of B. subtilis, which is condition-dependent, and the importance of evaluating probiotic strains under conditions relevant to the intended use.

Impaired glucose metabolism by deleting the operon of hydrogenase 2 in Escherichia coli.

Although Escherichia coli has four hydrogenases, their definite roles in fermentation are still not clear. In this study, all the operon deletion mutants of E.coli hydrogenases (∆hya, ∆hyb, ∆hyc, or ∆hyf) were constructed to evaluate the hydrogen metabolism in comparison to their respective single-gene deletion mutants of large subunits (∆hyaB, ∆hybC, ∆hycE, and ∆hyfG). Besides the hyc operon mutant that expectedly showed no hydrogen synthesis, the hyb operon mutant showed low hydrogen production and demonstrated significantly reduced growth under anaerobic conditions. The present work also provided first-hand data where deleterious effects of operon deletion were compared with single-gene deletion mutations and the results showed that the former type of deletion was found to cause more prominent phenotypic effects than the latterone. Interestingly, hyb operon mutant was remarkably distinct from other operon mutants, specifically in its inability to utilize glucose under both aerobic and anaerobic conditions. Further studies on this mutant revealed a significant reduction of the total intracellular ATP and NADH concentrations, which could explain its impaired glucose metabolism. In this way, Hyd-2 was verified as crucial not only in glucose metabolism but also in energy balance and redox homeostasis of the cells. Furthermore, a decreased expression of glucose metabolism-associated genes, particularly ppc and pykA, indicated their regulation by hyb operon, and thereby, glucose consumption. Moreover, the transcriptional changes in this mutant indicated the wide genomic connectivity of hyb operon to other metabolisms.

Salmonella Central Carbon Metabolism Enhances Bactericidal Killing by Fluoroquinolone Antibiotics.

The efficacy of killing by bactericidal antibiotics has been reported to depend in large part on the ATP levels, with low levels of ATP leading to increased persistence after antibiotic challenge. Here, we show that an atp operon deletion strain of Salmonella enterica serovar Typhimurium lacking the ATP synthase was at least 10-fold more sensitive to killing by the fluoroquinolone antibiotic ciprofloxacin and yet showed either increased survival or no significant difference compared with the wild-type strain when challenged with aminoglycoside or β-lactam antibiotics, respectively. The increased cell killing and reduced bacterial survival (persistence) after fluoroquinolone challenge were found to involve metabolic compensation for the loss of the ATP synthase through central carbon metabolism reactions and increased NAD(P)H levels. We conclude that the intracellular ATP levels per se do not correlate with bactericidal antibiotic persistence to fluoroquinolone killing; rather, the central carbon metabolic pathways active at the time of challenge and the intracellular target of the antibiotic determine the efficacy of treatment.

Disruption of the mce operon from Streptomyces affects spore resistance and results in precocious germination

Streptomyces coelicoloris a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosisas an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Germination assays show that spores of Δmcegerminate earlier than WT S. coelicolor, and impression mounts indicate spore chains emerge earlier in the Δmcestrain. As spores of Δmce possess an altered spore coat, it was hypothesised they might show reduced resistance to stressors. Heat kill assays show that deletion of the mce operon results in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. Spores of the Δmcestrain are also less resistant to triclosan, and sulfobetaines. Preliminary results indicate that the mce operon of S. coelicolor may be a cholesterol importer, as Δmce shows reduced growth in the presence of cholesterol, compared to WT S. coelicolor.

The Involvement of Thiamine Uptake in the Virulence of Edwardsiella piscicida.

Edwardsiella piscicida is a pathogenic bacterium, which can infect a number of fish species and cause a disease termed edwardsiellosis, threatening global fish farming with high prevalence and mortality. Thiamine (Vitamin B1), functioning in the form of thiamine pyrophosphate (TPP), is essential for almost all organisms. Bacteria acquire TPP by biosynthesis or by transportation of exogenous thiamine. TPP availability has been associated with bacterial pathogenicity, but the underlying mechanisms remain to be discovered. The role of thiamine in the pathogenicity of E. piscicida is unknown. In this study, we characterized a thiamine transporter (TT) operon in E. piscicida. The deletion of the TT operon resulted in an intracellular TPP lacking situation, which led to attenuated overall pathogenicity, impaired abilities associated with motility and host cell adhesion, as well as decreased expression of certain flagellar and adhesion genes. Moreover, TPP starvation led to intracellular c-di-GMP reduction, and introducing into the TPP-suppressed mutant strain an exogenous diguanylate cyclase for c-di-GMP synthesis restored the virulence loss. Taken together, this work reveals the involvement of thiamine uptake in the virulence regulation of E. piscicida, with c-di-GMP implicated in the process. These finding could be employed to explore potential drug targets against E. piscicida.

Bacteriophage S6 requires bacterial cellulose for Erwinia amylovora infection.

Bacteriophages are highly selective in targeting bacteria. This selectivity relies on the specific adsorption of phages to the host cell surface. In this study, a Tn5 transposon mutant library of Erwinia amylovora, the causative agent of fire blight, was screened to identify bacterial receptors required for infection by the podovirus S6. Phage S6 was unable to infect mutants with defects in the bacterial cellulose synthase operon (bcs). The Bcs complex produces and secretes bacterial cellulose, an extracellular polysaccharide associated with bacterial biofilms. Deletion of the bcs operon or associated genes (bcsA, bcsC and bcsZ) verified the crucial role of bacterial cellulose for S6 infection. Application of the cellulose binding dye Congo Red blocked infection by S6. We demonstrate that infective S6 virions degraded cellulose and that Gp95, a phage-encoded cellulase, is involved to catalyse the reaction. In planta S6 did not significantly inhibit fire blight symptom development. Moreover, deletion of bcs genes in E. amylovora did not affect bacterial virulence in blossom infections, indicating that sole application of cellulose targeting phages is less appropriate to biologically control E. amylovora. The interplay between cellulose synthesis, host cell infection and maintenance of the host cell population is discussed.

Acid resistance system CadBA is implicated in acid tolerance and biofilm formation and is identified as a new virulence factor of Edwardsiella tarda

Edwardsiella tarda is a facultative intracellular pathogen in humans and animals. The Gram-negative bacterium is widely considered a potentially important bacterial pathogen. Adaptation to acid stress is important for the transmission of intestinal microbes, so the acid-resistance (AR) system is essential. However, the AR systems of E. tarda are totally unknown. In this study, a lysine-dependent acid resistance (LDAR) system in E. tarda, CadBA, was characterized and identified. CadB is a membrane protein and shares high homology with the lysine/cadaverine antiporter. CadA contains a PLP-binding core domain and a pyridoxal phosphate-binding motif. It shares high homology with lysine decarboxylase. cadB and cadA are co-transcribed under one operon. To study the function of the cadBA operon, isogenic cadA, cadB and cadBA deletion mutant strains TX01ΔcadA, TX01ΔcadB and TX01ΔcadBA were constructed. When cultured under normal conditions, the wild type strain and three mutants exhibited the same growth performance. However, when cultured under acid conditions, the growth of three mutants, especially TX01ΔcadA, were obviously retarded, compared to the wild strain TX01, which indicates the important involvement of the cadBA operon in acid resistance. The deletion of cadB or cadA, especially cadBA, significantly attenuated bacterial activity of lysine decarboxylase, suggesting the vital participation of cadBA operon in lysine metabolism, which is closely related to acid resistance. The mutations of cadBA operon enhanced bacterial biofilm formation, especially under acid conditions. The deletions of the cadBA operon reduced bacterial adhesion and invasion to Hela cells. Consistently, the deficiency of cadBA operon abated bacterial survival and replication in macrophages, and decreased bacterial dissemination in fish tissues. Our results also show that the expression of cadBA operon and regulator cadC were up-regulated upon acid stress, and CadC rigorously regulated the expression of cadBA operon, especially under acid conditions. These findings demonstrate that the AR CadBA system was a requisite for the resistance of E. tarda against acid stress, and played a critical role in bacterial infection of host cells and in host tissues. This is the first study about the acid resistance system of E. tarda and provides new insights into the acid-resistance mechanism and pathogenesis of E. tarda.

Yersinia pestis Lipopolysaccharide Remodeling Confers Resistance to a Xenopsylla cheopis Cecropin.

Fleas are major vectors of Yersinia pestis, the causative agent of plague. It has been proposed that Y. pestis has developed the ability to overcome the innate immune responses of fleas. Despite the fact that they transmit a number of bacterial infections, very little is known about the immune responses in fleas. In this study, we describe the antimicrobial activities of a cecropin from Xenopsylla cheopis (cheopin), an efficient vector for Y. pestis in the wild. This is the first cecropin-class antimicrobial peptide described from Siphonaptera insects. Cheopin showed potent activity against Gram-negative bacteria but little activity against wild-type Y. pestis KIM6+. Deletion of the aminoarabinose operon, which is responsible for the 4-amino-4-deoxy-l-arabinose (Ara4N) modification of LPS, rendered Y. pestis highly susceptible to cheopin. Confocal microscopy and whole cell binding assays indicated that Ara4N modification reduces the affinity of cheopin for Y. pestis. Further, cheopin only permeabilized bacterial membranes in the absence of Ara4N-modified LPS, which was correlated with bacterial killing. This study provides insights into innate immunity of the flea and evidence for the crucial role of Ara4N modification of Y. pestis LPS in conferring resistance against flea antimicrobial peptides.

Deletion of the PA4427-PA4431 Operon of Pseudomonas aeruginosa PAO1 Increased Antibiotics Resistance and Reduced Virulence and Pathogenicity by Affecting Quorum Sensing and Iron Uptake.

The respiratory chain is very important for bacterial survival and pathogenicity, yet the roles of the respiratory chain in P. aeruginosa remain to be fully elucidated. Here, we not only proved experimentally that the operon PA4427-PA4431 of Pseudomonas aeruginosa PAO1 encodes respiratory chain complex III (cytobc1), but also found that it played important roles in virulence and pathogenicity. PA4429–31 deletion reduced the production of the virulence factors, including pyocyanin, rhamnolipids, elastase, and extracellular polysaccharides, and it resulted in a remarkable decrease in pathogenicity, as demonstrated in the cabbage and Drosophila melanogaster infection models. Furthermore, RNA-seq analysis showed that PA4429–31 deletion affected the expression levels of the genes related to quorum-sensing systems and the transport of iron ions, and the iron content was also reduced in the mutant strain. Taken together, we comprehensively illustrated the function of the operon PA4427–31 and its application potential as a treatment target in P. aeruginosa infection.

The msaABCR Operon Regulates Persister Formation by Modulating Energy Metabolism in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that causes chronic, systemic infections, and the recalcitrance of these infections is mainly due to the presence of persister cells, which are a bacterial subpopulation that exhibits extreme, yet transient, antibiotic tolerance accompanied by a transient halt in growth. However, upon cessation of antibiotic treatment, a resumption in growth of persister cells causes recurrence of infections and treatment failure. Previously, we reported the involvement of msaABCR in several important staphylococcal phenotypes, including the formation of persister cells. Additionally, observations of the regulation of several metabolic genes by the msaABCR operon in transcriptomics and proteomics analyses have suggested its role in the metabolic activities of S. aureus. Given the importance of metabolism in persister formation as our starting point, in this study we demonstrated how the msaABCR operon regulates energy metabolism and subsequent antibiotic tolerance. We showed that deletion of the msaABCR operon results in increased tricarboxylic acid (TCA) cycle activity, accompanied by increased cellular ATP content and higher NADH content in S. aureus cells. We also showed that msaABCR (through MsaB) represses the ccpE and ndh2 genes, thereby regulating TCA cycle activity and the generation of membrane potential, respectively. Together, the observations from this study led to the conclusion that msaABCR operon deletion induces a metabolically hyperactive state, leading to decreased persister formation in S. aureus.

#17: DNA Methylation in Streptococcus pyogenes Promotes M-Protein Expression and Enhances Epithelial Cell Adherence and Persistence on a Mucosal Surface

Abstract Background DNA methylation has been extensively studied as a regulator of gene expression among eukaryotes, but the regulatory role for DNA methylation has been far less studied in bacterial pathogens. Streptococcus pyogenes, or Group A Streptococcus, is an important bacterial pathogen of children. Our group has recently shown that S. pyogenes utilizes DNA methylation at N6-methyladenine (m6A) as a regulatory mechanism, modulating gene transcription and influencing the expression of several genes recognized as potential virulence factors. Our goal was to further explore how DNA methylation impacts virulence properties of S. pyogenes through adherence to epithelial cells and persistence on a mucosal surface. Methods S. pyogenes strains HSC12 (M14), MEW123 (M28), and MEW431 (M4) were modified by in-frame genetic deletion of a 3-gene operon encoding the only Type-I Restriction-Modification locus, resulting in mutant strains lacking the majority of m6A base modifications (ΔRSM). S. pyogenes parent and ΔRSM mutant strains were subjected to transcriptional profiling by RT–PCR and RNA-Seq analysis. Adherence rates of streptococci to D562 human pharyngeal epithelial cells and VKE6E7 human vaginal epithelial cells were assessed. A murine vaginal mucosa colonization model was used to monitor streptococcal mucosal persistence. Results The ΔRSM mutants of all three strains lacked essentially all m6A DNA base modifications by dot-blot with anti-m6A antibody and PacBio™ sequencing with methylation analysis. Transcriptional profiling demonstrated that a limited subset of ~20 genes was strongly down-regulated in all of the ΔRSM mutant strains, most notably genes in the core Mga regulon involved in tissue adherence and evasion of the host immune response, including the M protein (emm gene). The ΔRSM mutants of all 3 strains were attenuated for adherence to human respiratory and vaginal epithelial cells compared with parent strains or complemented mutant strains. The HSC12 and MEW431 ΔRSM mutant strains exhibited significantly decreased bacterial burdens over time compared with parent strains in the murine mucosal carriage model. The bacterial burdens of strain MEW123 and its ΔRSM mutant were not significantly different in the murine mucosal carriage model. Expression of R28, an adhesin specifically promoting adherence to vaginal epithelial cells, was not altered in the MEW123 ΔRSM mutant, which may explain the continued persistence of this strain in the murine model. Conclusions DNA methylation influences gene expression at the transcriptional level in S. pyogenes and affects virulence properties in both in vitro and in vivo models of infection. We report that methylation promotes activation of several important virulence factors, including the M protein and other members of the Mga regulon, and influences epithelial cell adherence and streptococcal persistence on a mucosal surface. DNA methylation appears to be an important contributor to bacterial physiology and pathogenesis. Future work will identify the interaction of m6A base modifications and transcriptional regulatory proteins.

Post-translational knockdown and post-secretional modification of EsxA determine contribution of EsxA membrane permeabilizing activity for mycobacterial intracellular survival

ABSTRACT Current genetic studies (e.g. gene knockout) have suggested that EsxA and EsxB function as secreted virulence factors that are essential for Mycobaterium tuberculosis (Mtb) intracellular survival, specifically in mediating phagosome rupture and translocation of Mtb to the cytosol of host cells, which further facilitates Mtb intracellular replicating and cell-to-cell spreading. The EsxA-mediated intracellular survival is presumably achieved by its pH-dependent membrane-permeabilizing activity (MPA). However, the data from other studies have generated a discrepancy regarding the role of EsxA MPA in mycobacterial intracellular survival, which has raised a concern that genetic manipulations, such as deletion of esxB-esxA operon or RD-1 locus, may affect other codependently secreted factors that could be also directly involved cytosolic translocation, or stimulate extended disturbance on other genes’ expression. To avoid the drawbacks of gene knockout, we first engineered a Mycobacterium marinum (Mm) strain, in which a DAS4+ tag was fused to the C-terminus of EsxB to allow inducible knockdown of EsxB (also EsxA) at the post-translational level. We also engineered an Mm strain by fusing a SpyTag (ST) to the C-terminus of EsxA, which allowed inhibition of EsxA-ST MPA at the post-secretional level through a covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and functional inhibition of EsxA resulted in attenuation of Mm intracellular survival in lung epithelial cells or macrophages, which unambiguously confirms the direct role of EsxA MPA in mycobacterial intracellular survival.

The role of the msaABCR operon in implant-associated chronic osteomyelitis in Staphylococcus aureus USA300 LAC

BackgroundThe msaABCR operon regulates several staphylococcal phenotypes such as biofilm formation, capsule production, protease production, pigmentation, antibiotic resistance, and persister cells formation. The msaABCR operon is required for maintaining the cell wall integrity via affecting peptidoglycan cross-linking. The msaABCR operon also plays a role in oxidative stress defense mechanism, which is required to facilitate persistent and recurrent staphylococcal infections. Staphylococcus aureus is the most frequent cause of chronic implant-associated osteomyelitis (OM). The CA-MRSA USA300 strains are predominant in the United States and cause severe infections, including bone and joint infections.ResultsThe USA300 LAC strain caused significant bone damage, as evidenced by the presence of severe bone necrosis with multiple foci of sequestra and large numbers of multinucleated osteoclasts. Intraosseous survival and biofilm formation on the K-wires by USA300 LAC strains was pronounced. However, the msaABCR deletion mutant was attenuated. We observed minimal bone necrosis, with no evidence of intramedullary abscess and/or fibrosis, along reduced intraosseous bacterial population and significantly less biofilm formation on the K-wires by the msaABCR mutant. microCT analysis of infected bone showed significant bone loss and damage in the USA300 LAC and complemented strain, whereas the msaABCR mutant’s effect was reduced. In addition, we observed increased osteoblasts response and new bone formation around the K-wires in the bone infected by the msaABCR mutant. Whole-cell proteomics analysis of msaABCR mutant cells showed significant downregulation of proteins, cell adhesion factors, and virulence factors that interact with osteoblasts and are associated with chronic OM caused by S. aureus.ConclusionThis study showed that deletion of msaABCR operon in USA300 LAC strain lead to defective biofilm in K-wire implants, decreased intraosseous survival, and reduced cortical bone destruction. Thus, msaABCR plays a role in implant-associated chronic osteomyelitis by regulating extracellular proteases, cell adhesions factors and virulence factors. However additional studies are required to further define the contribution of msaABCR-regulated molecules in osteomyelitis pathogenesis.

Identification of bacteria-derived urease in the coral gastric cavity

Nitrogen cycle is critical to maintain a healthy coral reef ecosystem. Urea can provide a source of nitrogen for coral holobiont and is important for coral calcification through degradation by urease. Despite the involvement of coral-associated bacteria in nitrogen fixation, nitrification and denitrification are well recognized, to what extend urea degradation by bacteria contributing to the urea utilization by coral holobiont remains to be investigated. In this study, we demonstrate that the urea utilization is a common feature of Halomonas spp. that is a dominant genus in cultivable coral-associated bacteria. A urease operon was characterized by genome sequencing and gene knock-out technique in Halomonas meridiana SCSIO 43005, isolated from the gastric cavity of healthy scleractinian coral Galaxea fascicularis. H. meridiana showed high urease activity which was induced by urea and deletion of the urease operon reduced the capability to use urea as solo nitrogen source. Furthermore, approximately 1/3 coral-associated bacteria in the IMG/M database possess complete urease operons indicating the involvement of bacteria-derived ureases in coral holobiont. These results suggest that urease from coral-associated bacteria might be important player in the nitrogen cycling of coral reefs.

Enhancing β-Carotene Production in Escherichia coli by Perturbing Central Carbon Metabolism and Improving the NADPH Supply.

Beta (β)-carotene (C40H56; a provitamin) is a particularly important carotenoid for human health. Many studies have focused on engineering Escherichia coli as an efficient heterologous producer of β-carotene. Moreover, several strains with potential for use in the industrial production of this provitamin have already been constructed via different metabolic engineering strategies. In this study, we aimed to improve the β-carotene-producing capacity of our previously engineered E. coli strain ZF43ΔgdhA through further gene deletion and metabolic pathway manipulations. Deletion of the zwf gene increased the resultant strain's β-carotene production and content by 5.1 and 32.5%, respectively, relative to the values of strain ZF43ΔgdhA, but decreased the biomass by 26.2%. Deletion of the ptsHIcrr operon further increased the β-carotene production titer from 122.0 to 197.4 mg/L, but the provitamin content was decreased. Subsequently, comparative transcriptomic analysis was used to explore the dynamic transcriptional responses of the strains to the blockade of the pentose phosphate pathway and inactivation of the phosphotransferase system. Lastly, based on the analyses of comparative transcriptome and reduction cofactor, several strategies to increase the NADPH supply were evaluated for enhancement of the β-carotene content. The combination of yjgB gene deletion and nadK overexpression led to increased β-carotene production and content. The best strain, ECW4/p5C-nadK, produced 266.4 mg/L of β-carotene in flask culture and 2,579.1 mg/L in a 5-L bioreactor. The latter value is the highest reported from production via the methylerythritol phosphate pathway in E. coli. Although the strategies applied is routine in this study, the combinations reported were first implemented, are simple but efficient and will be helpful for the production of many other natural products, especially isoprenoids. Importantly, we demonstrated that the use of the methylerythritol phosphate pathway alone for efficient β-carotene biosynthesis could be achieved via appropriate modifications of the cell metabolic functions.

Effect of deletion of gene cluster involved in synthesis of Enterobacterial common antigen on virulence and immunogenicity of live attenuated Salmonella vaccine when delivering heterologous Streptococcus pneumoniae antigen PspA

BackgroundEnterobacterial common antigen (ECA) is a family-specific surface antigen shared by all members of the Enterobacteriaceae family. Previous studies showed that the loss of ECA results in Salmonella attenuation, indicating its usefulness as a vaccine candidate for Salmonella infection, but no studies have shown whether the mutation resulting from the deletion of the ECA operon in conjunction with other mutations could be used as an antigen vehicle for heterologous protein antigen delivery.ResultsIn this study, we introduced a nonpolar, defined ECA operon deletion into wild-type S. Typhimurium χ3761 and an attenuated vaccine strain χ9241, obtaining two isogenic ECA operon mutants, namely, χ12357 and χ12358, respectively. A number of in vitro and in vivo properties of the mutants were analyzed. We found that the loss of ECA did not affect the growth, lipopolysaccharide (LPS) production and motility of S. Typhimurium wild type strain χ3761 and its attenuated vaccine strain χ9241 but significantly affected the virulence when administered orally to BALB/c mice. Furthermore, the effects of the ECA mutation on the immunogenicity of a recombinant S. Typhimurium vaccine strain χ9241 when delivering the pneumococcal antigen PspA were determined. The result showed that the total anti-PspA IgG level of χ12358 (pYA4088) was slightly lower than that of χ9241 (pYA4088), but the protection rate was not compromised.ConclusionsECA affects virulence and benefits the Th2 immunity of Salmonella Typhimurium, therefore, it is feasible to use a reversible ECA mutant mode to design future Salmonella vaccine strains for heterologous protective antigens.

Emergence of a clone of invasive fucK-negative serotype e Haemophilus influenzae in British Columbia.

Introduction of the Haemophilus influenzae serotype b (Hib) conjugate vaccine has changed the epidemiology of invasive H. influenzae disease, with most infections now caused by non-typeable (non-encapsulated) and non-Hib encapsulated strains. We describe nine invasive serotype e H. influenzae (Hie) from British Columbia that were determined to have complete deletion of their fucose operon genes. These nine isolates were recovered from blood cultures of three female and six male patients during 2011-2018, with eight recovered in the past 4 years. All nine isolates were biotype IV, with eight showing identical pulsed field gel electrophoresis (PFGE) profiles, whereas the ninth showed 95% similarity. PFGE analysis also showed these fucose operon-negative Hie to be most (94%) similar to the multi-locus sequence type (ST)-18, the most common ST among Hie in British Columbia. These nine fucose operon-negative Hie represented 27.3% of the 33 invasive Hie isolated in British Columbia from 2010 to 2018. Deletion of the fucose operon did not appear to impact the transmission ability of these strains or their ability to cause invasive disease.