Selected Topics from Classical Bacterial Genetics

Raleigh, Elisabeth A.; Elbing, Karen L.; Brent, Roger

doi:10.1002/0471142727.mb0104s59

Cited by 25 publications

(26 citation statements)

References 45 publications

(29 reference statements)

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“…Restriction and modification enzymes were purchased from Fermentas (Ontario, Canada) and were used according to the manufacturer's instructions. All DNA manipulations were performed with E. coli DH5␣ (45,57). Plasmid DNA was isolated with the Wizard Plus SV plasmid miniprep kit (Promega).…”

Section: Methodsmentioning

confidence: 99%

In Vivo Analysis of Cobinamide Salvaging inRhodobacter sphaeroidesStrain 2.4.1

Gray

Escalante‐Semerena

2009

J Bacteriol

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The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B 12 ). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ ؉ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.

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Section: Methodsmentioning

confidence: 99%

In Vivo Analysis of Cobinamide Salvaging inRhodobacter sphaeroidesStrain 2.4.1

Gray

Escalante‐Semerena

2009

J Bacteriol

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“…DbamE cells displayed enhanced sensitivity to the anionic detergents deoxycholate and SDS, but not to the nonionic detergents Tween 20 and Triton X-100 (Table 2). Growth of DbamE cells was selectively inhibited by 0.5 M EDTA and by the antibiotics nalidixic acid, carbenicillin and rifampicin (Table 2), which target DNA gyrase, peptidoglycan transpeptidation and RNA polymerase, respectively (Raleigh et al, 2002). Hypersensitivity to each chemical was complemented by pbamE ( Table 2).…”

Section: Heat and Chemical Sensitivity Of The Dbame Mutantmentioning

confidence: 99%

The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane β-barrel proteins in Caulobacter crescentus

2010

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The outer membrane of Gram-negative bacteria is an essential compartment containing a specific complement of lipids and proteins that constitute a protective, selective permeability barrier. Outer membrane β-barrel proteins are assembled into the membrane by the essential hetero-oligomeric BAM complex, which contains the lipoprotein BamE. We have identified a homologue of BamE, encoded by CC1365, which is located in the outer membrane of the stalked alpha-proteobacterium Caulobacter crescentus. BamE associates with proteins whose homologues in other bacteria are known to participate in outer membrane protein assembly: BamA (CC1915), BamB (CC1653) and BamD (CC1984). Caulobacter cells lacking BamE grow slowly in rich medium and are hypersensitive to anionic detergents, some antibiotics and heat exposure, which suggest that the membrane integrity of the mutant is compromised. Membranes of the ΔbamE mutant have normal amounts of the outer membrane protein RsaF, a TolC homologue, but are deficient in CpaC*, an aggregated form of the outer membrane secretin for type IV pili. ΔbamE membranes also contain greatly reduced amounts of three TonB-dependent receptors that are abundant in wild-type cells. Cells lacking BamE have short stalks and are delayed in stalk outgrowth during the cell cycle. Based on these findings, we propose that Caulobacter BamE participates in the assembly of outer membrane β-barrel proteins, including one or more substrates required for the initiation of stalk biogenesis.

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“…DNA was extracted with phenol-chloroform, ethanol precipitated, and dissolved in 10 l of sequence loading buffer. After incubation at 95°C for 5 min, DNA was loaded onto a 6.5% (wt/vol) DNA sequencing gel (22). Appropriate sequencing reaction mixtures were loaded onto the gels along with the footprinting samples and used as a size ladder for identification of the sequences of protected sites.…”

mentioning

confidence: 99%

Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor

et al. 2010

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Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K D (equilibrium dissociation constant) of 15 M, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 M. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5-TGAAACCGGTTTCA-3. This palindrome overlaps with the RNA polymerase binding site.

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Selected Topics from Classical Bacterial Genetics

Cited by 25 publications

References 45 publications

In Vivo Analysis of Cobinamide Salvaging inRhodobacter sphaeroidesStrain 2.4.1

In Vivo Analysis of Cobinamide Salvaging inRhodobacter sphaeroidesStrain 2.4.1

The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane β-barrel proteins in Caulobacter crescentus

Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor

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