Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72?h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48?h. Even when the inoculation by P. putida was delayed for 24?h, P. fluorescens was eliminated from the capillary within 48?h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.     

Flagellin inhibits Myoviridae phage φCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.     

Extensive proteolysis of head and inner body proteins by a morphogenetic protease in the giant Pseudomonas aeruginosa phage φKZ

Encased within the 280 kb genome in the capsid of the giant myovirus φKZ is an unusual cylindrical proteinaceous 'inner body' of highly ordered structure. We present here mass spectrometry, bioinformatic and biochemical studies that reveal novel information about the φKZ head and the complex inner body. The identification of 39 cleavage sites in 19 φKZ head proteins indicates cleavage of many prohead proteins forms a major morphogenetic step in φKZ head maturation. The φKZ head protease, gp175, is newly identified here by a bioinformatics approach, as confirmed by a protein expression assay. Gp175 is distantly related to T4 gp21 and recognizes and cleaves head precursors at related but distinct S/A/G-X-E recognition sites. Within the φKZ head there are six high-copy-number proteins that are probable major components of the inner body. The molecular weights of five of these proteins are reduced 35-65% by cleavages making their mature form similar (26-31 kDa), while their precursors are dissimilar (36-88 kDa). Together the six abundant proteins sum to the estimated mass of the inner body (15-20 MDa). The identification of these proteins is important for future studies on the composition and function of the inner body.     

The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.     

The Escherichia coli rhamnose promoter rhaP BAD is in Pseudomonas putida KT2440 independent of Crp–cAMP activation

We developed an expression vector system based on the broad host range plasmid pBBR1MCS2 with the Escherichia coli rhamnose-inducible expression system for applications in Pseudomonas. For validation and comparison to E. coli, enhanced green fluorescent protein (eGFP) was used as a reporter. For further characterization, we also constructed plasmids containing different modifications of the rhaP _BAD promoter. Induction experiments after the successful transfer of these plasmids into Pseudomonas putida KT2440 wild-type and different knockout strains revealed significant differences. In Pseudomonas, we observed no catabolite repression of the rhaP _BAD promoter, and in contrast to E. coli, the binding of cyclic adenosine monophosphate (cAMP) receptor protein (Crp)–cAMP to this promoter is not necessary for induction as shown by deletion of the Crp binding site. The crp ⁻ mutant of P. putida KT2440 lacked eGFP expression, but this is likely due to problems in rhamnose uptake, since this defect was complemented by the insertion of the l-rhamnose-specific transporter rhaT into its genome via transposon mutagenesis. Other global regulators like Crc, PtsN, and CyoB had no or minor effects on rhamnose-induced eGFP expression. Therefore, this expression system may also be generally useful for Pseudomonas and other γ-proteobacteria.     

The Role of Cysteine 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putida

The cysteinyl residue at the active site of L-methionine γ-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k _cat toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the γ-elimination reaction of L-methionine and in substrate recognition in the MGLs.     

Salting-out extraction of recombinant κ-carrageenase and phage T7 released from Escherichia coli cells

Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI = 1 and release more than 90.0% of enzyme within 90 min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.     

The microbiota protects against Pseudomonas aeruginosa pneumonia via γδ T cell-neutrophil axis in mice

Exposure to antimicrobials leading to microbiota dysbiosis has been found to be an independent risk factor for extensively drug-resistant Pseudomonas aeruginosa acquisition. Microbiota dysbiosis may induce imbalanced immune responses and can affect disease susceptibility. However, the potential role of commensal microbiota in bacterial pneumonia is poorly defined. The aim of this study was to investigate the mechanistic basis for the defective host defenses against P. aeruginosa pneumonia induced by antibiotic pretreatment perturbing microbiota. We found that antibiotic pretreatment significantly perturbed the composition of intestinal microbiota. The microbiota dysbiosis impaired host defenses against P. aeruginosa pneumonia, as reflected by the increased bacterial burden and dissemination, compromised local inflammatory responses and shortened survival time in microbiota-depleted mice compared with controls. This impairment correlated with a defective γδ T17 cell and downstream neutrophil responses. Anti-TCRγδ-treated mice had changes similar to those in the microbiota-depleted mice. Overall, our results suggest the importance of microbiota in supporting the host defense against pneumonia, define a crucial role for the γδ T cell-neutrophil axis in the potential mechanism, and delineate the deleterious effects of antibiotic treatment on antibacterial defenses.     

The annotated complete DNA sequence of Enterococcus faecalis bacteriophage φEf11 and its comparison with all available phage and predicted prophage genomes

φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42,822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The genes were arranged in functional modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and possessed a predicted stem-loop structure in between predicted P(L) and P(R) promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers.     

A heat-stable nicotinamide–adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein

A thermostable NAD(P)⁺ glycohydrolase (EC 3.2.2.6) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis. A heat-labile inhibitor of the enzyme was also partially purified. Enzyme free of inhibitor is present in culture supernatants. After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions. The general properties of the enzyme and inhibitor are described. The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively. The binding of inhibitor and enzyme is inhibited by the presence of substrate.     

Conserved genomes of ΦKMV-like bacteriophages (T7 supergroup) active on Pseudomonas aeruginosa

The T7-like ΦKMV bacteriophage active on Pseudomonas aeruginosa was previously isolated by us and shown to have DNA resistant to many endonucleases. A loss of sensitive sites might be a consequence of a long ΦKMV evolution on different hosts. To elucidate, whether this trait is shared by other similar phages, several new ΦKMV-like phages were isolated from different sources and compared. All studied ΦKMV-like phages formed three groups, insignificantly differing in the number and localization of endonuclease-sensitive DNA sites. This confirms that the present-day phages of this species have highly conserved genomes. Mutational “restoration” of the lost sites may be restricted by a lethal effect. The ΦKMV-like phages were shown for the first time to increase the rate of in vitro accumulation of giant KZ-like phages of P. aeruginosa. This effect is characteristic only of ΦKMV-like phages.     

The Role of γc Cytokines (IL-2, IL-7, and IL-15) in Regulation of Activation-Induced Cell Death of Memory T Cells

Cell and Tissue Biology - The role of γc cytokines (IL-2, IL-7, and IL-15) in the regulation of apoptotic death of memory T cells under cultivation conditions in vitro was studied using the...     

The IL-7Rα pathway is quantitatively and functionally altered in CD8 T cells in multiple sclerosis

The IL-7Rα single nucleotide polymorphism rs6897932 is associated with an increased risk for multiple sclerosis (MS). IL-7Rα is a promising candidate to be involved in autoimmunity, because it regulates T cell homeostasis, proliferation, and antiapoptotic signaling. However, the exact underlying mechanisms in the pathogenesis of MS are poorly understood. We investigated whether CD4 and CD8 lymphocyte subsets differed in IL-7Rα expression and functionality in 78 MS patients compared with 59 healthy controls (HC). A significantly higher frequency of IL-7Rα(+) CD8 effector memory (CD8EM) was found in MS. Moreover, IL-7Rα membrane expression was significantly increased in MS in naive and memory CD8 (all p < 0.05) with a similar trend in CD8EM (p = 0.055). No correlation was found between the expression level or frequency of IL-7Rα(+)CD8(+) and rs6897932 risk allele carriership. Upon IL-7 stimulation, MS patients had stronger STAT5 activation in CD8EM compared with HC. IL-7 stimulation had a differential effect on both mRNA and protein expression of granzyme A and granzyme B between MS and HC. Stainings of different lesions in postmortem MS brain material showed expression of IL-7 and CD8(+)IL-7Rα(+) in preactive, but not in active, demyelinating MS lesions, indicating involvement of IL-7Rα(+) lymphocytes in lesion development. The intralesional production of IL-7 in combination with the lower threshold for IL-7-induced cytotoxicity in MS may enhance the pathogenicity of these CD8 T cells. This is of special interest in light of the established demyelinating and cytotoxic actions of granzyme A.     

The effect of the 3′ → 5′ exonuclease of T7 DNA polymerase on frameshifts and deletions

Both spontaneous frameshift mutation and deletion mutation were measured in a T7 phage deficient in the 3′ → 5′ exonuclease of T7 DNA polymerase. It was found that the absence of this exonuclease caused a marked increase in the revision of both plus one and minus one mutations. The exonuclease deficiency caused essentially no effect on the frequency of deletion between 10-bp direct repeats even when the segment between the direct repeats contained a 25-bp palindrome.     

TGF-β Sensitivity Restrains CD8+ T Cell Homeostatic Proliferation by Enforcing Sensitivity to IL-7 and IL-15

The pleiotropic cytokine TGF-β has been implicated in the regulation of numerous aspects of the immune response, including naïve T cell homeostasis. Previous studies found that impairing TGF-β responsiveness (through expression of a dominant-negative TGF-β RII [DNRII] transgene) leads to accumulation of memory phenotype CD8 T cells, and it was proposed that this resulted from enhanced IL-15 sensitivity. Here we show naïve DNRII CD8 T cells exhibit enhanced lymphopenia-driven proliferation and generation of “homeostatic” memory cells. However, this enhanced response occurred in the absence of IL-15 and, unexpectedly, even in the combined absence of IL-7 and IL-15, which were thought essential for CD8 T cell homeostatic expansion. DNRII transgenic CD8 T cells still require access to self Class I MHC for homeostatic proliferation, arguing against generalized dysregulation of homeostatic cues. These findings suggest TGF-β responsiveness is critical for enforcing sensitivity to homeostatic cytokines that limit maintenance and composition of the CD8 T cell pool. (154 words).