ALBERT

Notes: Abstract: The first step in the anaerobic pathway of benzoate degradation by Rhodopseudomonas palustris is catalyzed by benzoate-coenzyme A ligase. To study factors influencing the synthesis of this enzyme, a polyclonal antiserum was prepared and in immunoblot assays. Benzoate-CoA ligase was synthesized when cells were grown with benzoate, as well as with hydroxyl- and methyl-substituted benzoates. Partially reduced alicyclic compounds proposed to be intermediates in the benzoate pathway also induced benzoate-CoA ligase. Ligase synthesis was repressed by oxygen. The diversity of inducers is consistent with the observation that benzoate is a central intermediate in the degradation of a variety of aromatic acids with more complex structures.

Notes: In Escherichia coli and some other γ-Proteobacteria, the alternative σ factor RpoS functions as a regulator of the general stress response. The role of RpoS in Pseudomonas aeruginosa is not clear. Although P. aeruginosa RpoS contributes to the resistance to several environmental stresses, its role appears to be less pivotal than in E. coli. In P. aeruginosa, RpoS also regulates the production of several virulence factors and influences the expression of individual genes that are controlled by quorum sensing. Some quorum-controlled genes are induced by RpoS, whereas others are repressed. To gain insights about RpoS function in P. aeruginosa and to understand better the regulation of quorum-controlled genes, we used transcript profiling to define an RpoS regulon. We identified 772 genes regulated by RpoS in stationary but not in logarithmic growth phase (504 were induced and 268 were repressed), and we identified putative RpoS promoter sequence elements with similarity to the E. coli RpoS consensus in several of these genes. Many genes in the regulon, for example a set of chemotaxis genes, have assigned functions that are distinct from those in E. coli and are not obviously related to a stress response. Furthermore, RpoS affects the expression of more than 40% of all quorum-controlled genes identified in our previous transcriptome analysis. This highlights the significance of RpoS as a global factor that controls quorum-sensing gene expression at the onset of stationary phase. The transcription profiling results have allowed us to build a model that accommodates previous seemingly conflicting reports.

Notes: An aerotaxis gene, aer, was cloned from Pseudomonas putida PRS2000. A P. putida aer mutant displayed an altered aerotactic response in a capillary assay. Wild-type P. putida clustered at the air/liquid interface. In contrast, the aer mutant did not cluster at the interface, but instead formed a diffuse band at a distance from the meniscus. Wild-type aer, provided in trans, complemented the aer mutant to an aerotactic response that was stronger than wild-type. The P. putida Aer sequence is similar over its entire length to the aerotaxis (energy taxis) signal transducer protein, Aer, of Escherichia coli. The amino-terminus is similar to redox-sensing regulatory proteins, and the carboxy-terminus contains the highly conserved domain present in chemotactic transducers.

Notes: Pseudomonas putida is chemotactic to a range of organic compounds, including several aromatic compounds. Genes involved in this behavioral response were identified by Tn5 mutagenesis of P. putida PRS2000, resulting in a strain that was nonchemotactic to all chemoattractants tested. Cloning and sequencing of the DNA at the Tn5 insertion site revealed a 13-kb region that contained 12 open reading frames, 9 of which are homologous to chemotaxis, flagellar and motility genes in other bacterial species. This indicates that the basic chemotaxis machinery of P. putida is similar to that of other bacterial systems, even though some of the compounds that are sensed as attractants are different.

Notes: Abstract The beta-ketoadipate pathway is a chromosomally encoded convergent pathway for aromatic compound degradation that is widely distributed in soil bacteria and fungi. One branch converts protocatechuate, derived from phenolic compounds including p-cresol, 4-hydroxybenzoate and numerous lignin monomers, to beta-ketoadipate. The other branch converts catechol, generated from various aromatic hydrocarbons, amino aromatics, and lignin monomers, also to beta-ketoadipate. Two additional steps accomplish the conversion of beta-ketoadipate to tricarboxylic acid cycle intermediates. Enzyme studies and amino acid sequence data indicate that the pathway is highly conserved in diverse bacteria, including Pseudomonas putida, Acinetobacter calcoaceticus, Agrobacterium tumefaciens, Rhodococcus erythropolis, and many others. The catechol branch of the beta-ketoadipate pathway appears to be the evolutionary precursor for portions of the plasmid-borne ortho-pathways for chlorocatechol degradation. However, accumulating evidence points to an independent and convergent evolutionary origin for the eukaryotic beta-ketoadipate pathway. In the face of enzyme conservation, the beta-ketoadipate pathway exhibits many permutations in different bacterial groups with respect to enzyme distribution (isozymes, points of branch convergence), regulation (inducing metabolites, regulatory proteins), and gene organization. Diversity is also evident in the behavioral responses of different bacteria to beta-ketoadipate pathway-associated aromatic compounds. The presence and versatility of transport systems encoded by beta-ketoadipate pathway regulons is just beginning to be explored in various microbial groups. It appears that in the course of evolution, natural selection has caused the beta-ketoadipate pathway to assume a characteristic set of features or identity in different bacteria. Presumably such identities have been shaped to optimally serve the diverse lifestyles of bacteria.

Notes: Aromatic compounds are important growth substrates for microorganisms. They form a large group of diverse compounds including lignin monomers, amino acids, quinones, and flavonoids. Aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen which is essential for the hydroxylation and cleavage of aromatic ring structures. The anaerobic metabolism of low molecular mass soluble aromatic compounds requires, of necessity, a quite different strategy. In most known cases, aromaticity is broken by reduction and the ring is subsequently opened hydrolytically. A small number of different central aromatic intermediates can be reduced, the most common of which is benzoyl-CoA, a compound that is formed as a central intermediate in the degradation of a large number of aromatic growth substrates. This review concentrates on the anaerobic aromatic metabolism via the benzoyl-CoA pathway. The peripheral pathways that transform growth substrates to benzoyl-CoA include various types of novel reactions, for example carboxylation of phenolic compounds, reductive elimination of ring substituents like hydroxyl or amino groups, oxidation of methyl substituents, O-demethylation reactions and shortening of aliphatic side chains. The central benzoyl-CoA pathway differs in several aspects in the denitrifying, phototrophic and fermenting bacteria studied. In denitrifying and phototrophic bacteria it starts with the two-electron reduction of benzoyl-CoA to a cyclic dienoyl-CoA driven by the hydrolysis of two molecules of ATP to ADP+Pi. This ring reduction is catalyzed by benzoyl-CoA reductase and requires a low-potential ferredoxin as an electron donor. In Rhodopseudomonas palustris the cyclic diene is further reduced to cyclohex-1-ene-1-carboxyl-CoA. In the denitrifying species Thauera aromatica, the cyclic diene is hydrated to give 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Subsequent β-oxidation results in the formation of a cyclic β-oxo compound, followed by hydrolytic carbon ring opening yielding 3-hydroxypimelyl-CoA in the case of T. aromatica and pimelyl-CoA in the case of R. palustris. These intermediates are further β-oxidized via glutaryl-CoA; final products are 3 acetyl-CoA and 1 CO2. In fermenting bacteria benzoyl-CoA may possibly be reduced to the level of cyclohex-1-ene-1-carboxyl-CoA in an ATP-independent reaction. The genes coding for the enzymes of the central benzoyl-CoA pathway have been cloned and sequenced from R. palustris, T. aromatica, and Azoarcus evansii. Sequence analyses of the genes support the concept that phototrophic and denitrifying bacteria use two slightly different pathways to metabolize benzoyl-CoA. The gene sequences have in some cases been very helpful for the identification of possible catalytic mechanisms that were not obvious from initial characterizations of purified enzymes.

Notes: Rhodopseudomonas palustris strain RCB100 degrades 3-chlorobenzoate (3-CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3-CBA and determined its N-terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate-CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3-CBA as a sole carbon source. The aliA gene from the 3-CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His-tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3-CBA degrading strain was 10-fold more active with 3-CBA (kcat/Km of 4.3 × 104 M−1 s−1) than the enzyme from the sequenced strain (kcat/Km 0.32 × 104 M−1 s−1). The CGA009 enzyme was not sufficiently active with 3-CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.

Notes: [Auszug] Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant–derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe ...