Absence Of L-arabinose Research Articles

Salmonella Enteritidis ghost vaccine carrying the hemagglutinin globular head (HA1) domain from H1N1 virus protects against salmonellosis and influenza in chickens

This study evaluated the attenuated Salmonella Enteritidis (SE) ghost strain JOL2114 (Δlon ΔcpxR Δasd), which displays on the bacterial surface the H1N1 hemagglutinin globular head portion (HA1; amino acid residues 63-286) on the bacterial surface for protective efficacy against Salmonella and H1N1 challenge in the chicken model, as the birds are the predominant reservoirs for both diseases. The ghost system enhanced the lysis process by converging two lysis processes found in bacteriophages: bacteriophage PhiX174 lysis gene E and holin-endolysin genes found in bacteriophage λ, complemented with accessory lysis-related proteins Rz/Rz1. The present lysis machinery resulted in complete lysis of host-attenuated SE strains in about 24 hrs of incubation under a non-permissible temperature of 42 °C in the absence of L-arabinose, an antisense inducer that blocks lysis gene expression during the growth phase. SE ghost JOL2114 surface display of HA1 was confirmed by Western blot analysis resulting in an immune-reactive band of 31 kDa in size. Chicken immunization via intramuscular and oral routes yielded both SE and HA1 antigen-specific immune responses. Protective humoral and cell-mediated immune responses were effectively elicited against both Salmonella and influenza challenge. This efficient strategy of ghost generation employs a dual system of phage lysis for biological generation of SE ghosts that preserves the surface antigenic architecture, offering a rapid and effective way to generate vaccines that could be deployed in urgent circumstances to protect against both Salmonella and influenza infection.

Salmonella enterica serovar Enteritidis ghosts displaying a surface FliC adjuvant elicit a robust immune response and effective protection against virulent challenge

Salmonella Enteritidis (SE) is one of the most common culprits of foodborne disease in humans due to its horizontal transmission from infected animals to humans. The development of a safe vaccine against Salmonella would be important for both farm animals and humans concerning disease containment. The SE ghosts carrying FliC were genetically constructed using a special ghost plasmid pJHL184 that co-expressed FliC and the phage lysis gene E. These SE ghosts were characterized by ghost generation efficacy by increasing the culture temperature to “42 °C” in the absence of L-arabinose. This temperature change led to an ghost generation with almost complete lysis of the SE host strain in 48 hs. The expression of FliC was confirmed by Western blot analysis. Also, indirect ELISA was used to prove FliC specific antibody generation in immunized mice. The parenteral adjuvant effect of the FliC antigen was demonstrated by immunizing mice with pJHL184::flC, pJHL184 alone, or PBS alone. The mice were intramuscularly immunized at six weeks of age (n = 8) and boosted after three weeks of primary inoculation. A total of 32 mice were equally divided into four groups. Each group was treated with pJHL-ghosts alone, ghost surface displaying FliC adjuvant, and compared to the PBS and naïve control groups. The immunized mice demonstrated greater IgG and IgA antibody responses than did the PBS control group. Furthermore, the addition of the ghosts to the FliC led to a significant increase in both the humoral and cell-mediated immune responses compared to those in the ghost alone group. Besides, the in vitro antigen uptake and presentation studies revealed efficient antigen presentation on the mouse macrophage cell surfaces. This finding further corroborated the potential efficacy of immune stimulation by SE ghosts. After the virulent challenge, we observed a significant reduction in the bacterial load in the spleen and liver tissues in SE ghosts surface, displaying FliC adjuvant. Our results demonstrate a safe and effective strategy to prevent salmonellosis. They also suggest that the surface expression of flagellin (FliC) significantly enhances antigen-specific humoral and cell-mediated immune responses. This FliC expression can also enhance the protective efficacy of the bacterial ghosts-based vaccine against virulent challenge.

Effects of combinatorial expression of selA, selB and selC genes on the efficiency of selenocysteine incorporation in Escherichia coli

In Escherichia coli, four gene products(selA, selB, selC and selD) and a selenocysteine(Sec) insertion sequence( SECIS) are required for the correct translation of UGA codons encoding Sec. Previous studies have shown that the stoichiometry of selenoproteine mRNA and elongation factor SelB affect the efficiency of Sec incorporation. Herein lies a detailed analysis of the effects of co-expressing selA, selB and selC genes under inducible promoters on the incorporation efficiency of Sec. Over-expression of either selA or selB reduced the efficiency of Sec incorporation by 61.1% or 11.6%, respectively, compared to the over-expression of the reporter vector alone did. Concomitant over-expression of selC with selA or selB completely reversed the reduce of the efficiency of Sec but still reduced the efficiency of the incorporation relative to that observed for expression of selC alone. Over-expression of selC gene alone under L-arabinose induction reduced the efficiency of the incorporation relative to that observed for co-expressing selC with selA and selB under the control of its endogenous promoter in the absence of L-arabinose. Co-expression of selA, selB and selC with selA or selB under the control of inducible promoters increased the efficiency of Sec incorporation by 69.7%. Moreover, inducing selenoprotein-related gene expression during the late exponential phase increased the efficiency of Sec incorporation by a factor of 5.4 relative to that observed for the reporter vector alone. These results suggest that the co-expression of selA, selB and selC in Escherichia coli under the control of inducible promoters is a viable and promising strategy for increasing the yields of selenoproteins.

Gain of Function Mutations in Membrane Region M2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus

KtrB, the K(+)-translocating subunit of the Na(+)-dependent bacterial K(+) uptake system KtrAB, consists of four M(1)PM(2) domains, in which M(1) and M(2) are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M(2C) is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M(2C1) and M(2C3), which are connected by a nonhelical M(2C2) region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M(2C2) led to a huge gain of function of K(+) uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in V(max) for K(+) transport. Na(+) translocation by KtrB was not affected. Partial to complete deletions of M(2C2) also led to enhanced V(max) values for K(+) uptake via KtrB. However, several deletion variants also exhibited higher K(m) values for K(+) uptake and at least one deletion variant, KtrB(Delta326-328), also transported Na(+) faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M(2C2) forms a flexible structure within the membrane allowing M(2C3) to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M(2C2) forms a flexible gate controlling K(+) translocation at the cytoplasmic side of KtrB, and (ii) that M(2C2) is required for the interaction between KtrA and KtrB.

Identification and functional analysis of the gene cluster for L-arabinose utilization in Corynebacterium glutamicum.

Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter(-1)) but not at a higher concentration of l-arabinose (40 g liter(-1)). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter

We have developed a system for the induction of marker-free mutation of Bacillus subtilis. The system features both the advantages of the use of antibiotic-resistance markers for mutant selection, and the ability to efficiently remove the markers, leaving unmarked mutations in the genome. It utilizes both a selective marker cassette and a counter-selective marker cassette. The selective marker cassette contains a chloramphenicol-resistance gene and the araR gene, which encodes the repressor for the arabinose operon (ara) of B. subtilis. The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene (neo) fused to the ara promoter. First, the chromosomal araR locus is replaced with the counter-selective marker cassette by double-crossover homologous recombination and positive selection for Nm resistance. The selective marker cassette is connected with upstream and downstream sequences from the target locus, and is integrated into the upstream region of the target locus by a double-crossover event. This integration is also positively selected for, using chloramphenicol resistance. In the resultant strain, AraR, encoded by araR on the selective marker cassette, represses the expression of neo in the absence of l-arabinose. Finally, the eviction of the selective marker cassette together with the target locus is achieved by an intra-genomic single-crossover event between the two downstream regions of the target locus, and can be selected for based on Nm resistance, because of the excision of araR. The counter-selective marker cassette remaining in the genome, whose expression is switched on or off based on the excision or introduction of the selective marker cassette, is used again for the next round of deletion. Using this system, the 3.8 kb iolS-csbC region and the 41.8 kb hutM-csbC region have been efficiently and successfully deleted, without leaving markers in the target loci. The positive selection and simple procedure will make it a useful tool for the construction of multiple mutations.

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.

Cloning, functional analysis, and transcriptional regulation of the Bacillus subtilis araE gene involved in L-arabinose utilization.

The Bacillus subtilis araR locus (mapped at about 294 degrees on the genetic map) comprises two open reading frames with divergently arranged promoters, the regulatory gene, araR, encoding a repressor, and a partially cloned gene, termed araE by analogy to the Escherichia coli L-arabinose permease gene. Here, we report the cloning and sequencing of the entire araE gene encoding a 50.4-kDa polypeptide. The araE gene is monocistronic (as determined by Northern blot analysis), and its putative product is very similar to a number of prokaryotic proton-linked monosaccharide transporters (the group I family of membrane transport proteins). Insertional inactivation of the araE gene leads to a conditional Ara- phenotype dependent on the concentration of L-arabinose in the medium. Therefore, we assume that araE encodes a permease involved in L-arabinose transport into the cell. The araE promoter region contains -10 and -35 regions (as determined by primer extension analysis) very similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigmaA, and the -35 region of the transcription start point for araE is located 2 bp from the -35 region of the araR gene. Transcriptional studies demonstrated that the expression from the araE promoter is induced by L-arabinose, repressed by glucose, and negatively regulated by AraR. These observations are consistent with a model according to which in the absence of L-arabinose, AraR binds to a site(s) within the araE/araR promoter, preventing transcription from the araE promoter and simultaneously limiting the frequency of initiation from its own promoter; the addition of L-arabinose will allow transcription from the araE promoter and increase the frequency of initiation from the araR promoter.

Structural Basis for Ligand-Regulated Oligomerization of AraC

The crystal structure of the arabinose-binding and dimerization domain of the Escherchia coli gene regulatory protein AraC was determined in the presence and absence of L-arabinose. The 1.5 angstrom structure of the arabinose-bound molecule shows that the protein adopts an unusual fold, binding sugar within a beta barrel and completely burying the arabinose with the amino-terminal arm of the protein. Dimer contacts in the presence of arabinose are mediated by an antiparallel coiled-coil. In the 2.8 angstrom structure of the uncomplexed protein, the amino-terminal arm is disordered, uncovering the sugar-binding pocket and allowing it to serve as an oligomerization interface. The ligand-gated oligomerization as seen in AraC provides the basis of a plausible mechanism for modulating the protein's DNA-looping properties.

How AraC Interacts Specifically with its Target DNAs

Previous work indicates that one subunit of the AraC protein dimer binds to a DNA target araI 1 , of 17 base-pairs. We systematically substituted every base-pair in a synthetic araI 1 target with the three possible alternatives and then tested binding of araI 1 and of these 51 DNA targets to AraC by quantitative gel shift analysis in the presence of L-arabinose. We found that every substitution of the underlined bases reduces AraC binding tenfold or more: 5′ T AGC ATTTTTA TCCATA 3′. Substitutions at other bases have little or no effect. In the absence of L-arabinose we observed a sixfold reduction of binding of AraC to araI 1 . We have designated the 5′ AGC sequence the A-box and the 5′ TCCATA sequence the B-box. We synthesised DNA targets containing either two A or two B-boxes with the natural araI 1 - I 2 spacing. Wild-type AraC binds both targets in the presence of L-arabinose in a gel shift experiment. In the absence of L-arabinose, AraC binds only to the double B-box. We then tested various AraC mutant proteins in the same way. S208A and H212A bind to the double B-box but not to the double A-box in the presence or absence of L-arabinose. D256A binds to the double A-box, but not to the double B-box, in the presence of L-arabinose but not in its absence. The implications of these results for the mechanism of AraC induction by L-arabinose are discussed.

Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r.

Mutations in the araC gene have been isolated which alter both the activator and autoregulatory functions of AraC protein (L.G. Cass and G. Wilcox, J. Bacteriol. 166:892-900, 1986). In this study, the effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which was not observed in the araC+ cell. Experiments were therefore focused on understanding the mechanism of this novel activation. We describe a systematic analysis of the effect of mutations within the known regulatory binding sites for araBAD and araC transcription on araC expression. Our results suggest that the novel activation of araC expression requires the AraC activator-binding site, araI, and the cyclic AMP receptor protein-cyclic AMP complex-binding site. We also found that in the absence of L-arabinose, the araI site was required for maximal autoregulation by the wild-type AraC protein.

Interaction of the regulatory gene product with the operator site in the l-arabinose operon of Escherichia coli

The DNA binding properties of the araC protein in the absence of l-arabinose have been studied in Escherichia coli using the nitrocellulose membrane filter technique. Equilibrium competition experiments demonstrate that the araC protein binds specifically to the ara operator. The apparent K m of the interaction is 1 × 10 −12 m at 20 °C. The rates of association and dissociation of the complex have also been determined. A k a of 2 × 10 9 m −1 s −1at 20 °C is calculated assuming binding to a single site. The half-life of the complex is three minutes. The equilibrium constant calculated from the ratio of k a to k d is 2.8 × 10 −12 m at 20 °C. The good agreement between the equilibrium and kinetic determinations of the equilibrium constant suggest that the kinetic studies are providing true rate constants. It is calculated that about 1% of the purified araC protein is active with respect to operator binding activity.

Dual control of arabinose genes on transducing phage λd ara

A λ-φ80 hybrid derivative of the defective transducing phage φ80d ara (Gottesman & Beckwith, 1969) has most of its late genes replaced by Escherichia coli DNA containing an intact arabinose operon. In a cell lysogenic for this phage the arabinose operon on the phage responds normally to its regulatory gene araC and to the level of l-arabinose in the growth medium. However, as a consequence of transcriptional read-through from phage late genes to the arabinose operon, arabinose enzymes are also synthesized under the control of phage gene Q during growth of the phage in the absence of l-arabinose. We also show that the gene araC has the same transcriptional orientation as the phage late genes and the arabinose operon genes B, A and D. Since monomers of phage lysozyme and l-arabinose isomerase are produced in comparable quantities under phage late control, it follows that if there is transcription termination at the end of the gene araC it is no more than 90% efficient.