Check out our New Publishers' Select for Free Articles

Archives of Microbiology (v.172, #5)

Microbial genomes and “missing” enzymes: redefining biochemical pathways by S. J. Cordwell (pp. 269-279).

A biochemical pathway is the representation of a defined set of substrates, enzyme reactions and products linked together to generate an outcome beneficial to a living cell. Microbial genome sequence data are unparalleled resources for understanding cellular metabolism without the prior definitions imposed by classical biochemistry. Simple analysis of three well-studied biochemical pathways (the tricarboxylic acid cycle, pentose phosphate pathway and glycolysis) from the 17 publicly available microbial genomes has shown that these pathways may rarely occur as previously defined. Therefore, following whole-genome sequencing it has become necessary to redefine the “classical” biochemical steps leading from substrate to end-product for each pathway. Often, unique or alternative reactions appear to be required in order to maintain pathway functionality where expected enzyme reactions (as defined by the presence or absence of the corresponding genes) are “missing”. Conversely, such enzymes may be accounted for by: (1) the presence of low sequence similarity or novel genes encoding enzymes performing the same or similar functions, (2) the presence of multienzyme proteins, (3) incorrectly assigned gene identities in genome databases, and (4) known enzyme functions that have yet to be correlated with a gene sequence. Most importantly, the presence of a gene sequence does not necessarily ensure that its corresponding enzyme is actually functional. This may be due to the presence of nonactive remnant genes, evolutionary pressures leading to loss of function, inactivating mutations, the lack of transcription/translation, and post-translational processing. Modifications at the gene and/or functional levels, as well as the possible use of alternative enzymes, must be considered when reconstructing biochemical pathways for fully sequenced microbial genomes.

Keywords: Keywords Functional genomics; Genome sequencing; Glycolysis; Microbial metabolism; Pentose phosphate pathway; Tricarboxylic acid cycle

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii by Roberto Zazueta-Sandoval; J. Félix Gutiérrez-Corona (pp. 280-286).

A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K _m values obtained for acetaldehyde, ethanol, NADH₂, and NAD⁺ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol.

Keywords: Key words Alcohol dehydrogenase; Mucor rouxii; Yeast/mold dimorphism; Developmental/environmental regulation

Anaerobic degradation of m-cresol by Desulfobacterium cetonicum is initiated by formation of 3-hydroxybenzylsuccinate by J. A. Müller; Alexander S. Galushko; Andreas Kappler; Bernhard Schink (pp. 287-294).

The anaerobic bacterium Desulfobacterium cetonicum oxidized m-cresol completely with sulfate as electron acceptor. During growth, 3-hydroxybenzylsuccinate (identified by gas chromatography/mass spectroscopy and by comparison of high-performance liquid chromatography retention time and UV spectrum with a chemically synthesized reference compound) accumulated in the medium. This finding indicates that the methyl group of m-cresol is activated by addition to fumarate as in the case of anaerobic toluene metabolism. In cell-free extracts of D. cetonicum, the formation of 3-hydroxybenzylsuccinate from m-cresol and fumarate was detected at an activity of 0.5 nmol min^–1 (mg protein)^–1. This reaction depended strictly on anoxic assay conditions. Treatment with air resulted in a complete loss of activity; however, some activity could be recovered after restoring anoxic conditions. The activity was slightly membrane-associated. 3-Hydroxybenzylsuccinate was degraded via CoA thioesterification and further oxidation to 3-hydroxybenzoyl-CoA as subsequent steps in the degradation pathway.

Keywords: Key words Anaerobic degradation; Aromatic; compounds; m-Cresol; Toluene; 3-Hydroxybenzylsuccinate; Sulfate-reducing bacteria; Desulfobacterium cetonicum

Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli by Iris Stallkamp; William Dowhan; Karlheinz Altendorf; K. Jung (pp. 295-302).

Synthesis of the high-affinity K⁺-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K⁺ limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K⁺ to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD.

Keywords: Key words Escherichia coli; K+; Turgor; Phospholipids; Histidine kinase

Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria by Gerda Harms; Ralf Rabus; F. Widdel (pp. 303-312).

The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud. Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks). Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months. Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively. Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., α-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids. Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions. Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene. Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack. Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1. Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the β-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis. This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes.

Keywords: Key words Anaerobic degradation; Denitrifying; bacteria; Aromatic hydrocarbons; Alkylbenzenes; p-Cymene; Alkylbenzoates; Intermediates; 16S rRNA sequence analysis

A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens by Francisco Martínez Murillo; Theresa Gugliuzza; John Senko; Partha Basu; J. F. Stolz (pp. 313-320).

Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated. Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction. The apparent K _mvalues for nitrate and nitrite were determined to be 32 and 10 μM, respectively. Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM. Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate. An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis. It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa. The 62-kDa polypeptide [a low-midpoint potential (–207 mV), multiheme cytochrome c] exhibited nitrite reductase activity under denaturing conditions. No molybdenum was detected in the complex by plasma-emission mass spectrometry.

Keywords: Key wordsGeobacter metallireducens; Nitrate; reduction; Nitrite reduction; Cytochrome c

Osmoadaptation in halophilic and alkaliphilic methanotrophs by Valentina N. Khmelenina; Marina G. Kalyuzhnaya; Valentin G. Sakharovsky; Natalia E. Suzina; Yuri A. Trotsenko; G. Gottschalk (pp. 321-329).

By using ¹H- and ¹³C-NMR spectroscopy, an accumulation of sucrose and two cyclic amino acids [ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) and 5-oxoproline (pyrrolidone carboxylic acid)] was detected in the halotolerant methanotrophs Methylobacter alcaliphilus 20Z and Methylobacter modestohalophilus 10S. The organic solute pool was found to increase upon raising the NaCl concentration. In M. alcaliphilus 20Z, the intracellular level of the total solutes was shown to be sufficient to balance the osmotic pressure of the medium, whereas in M. modestohalophilus 10S their content was several times lower. Additionally, phosphatidylglycerol and phosphatidylcholine were predominant cell phospholipids in salt-adapted M. alcaliphilus 20Z. However, no phosphatidylcholine was found in M. modestohalophilus 10S, and the portion of phosphatidylglycerol increased while phosphatidylethanolamine decreased upon elevated external NaCl concentrations. Regularly arranged glycoprotein surface layers (S-layers) of hexagonal and linear (p2) symmetry were observed on the outer cell walls of M. alcaliphilus 20Z and M. modestohalophilus 10S. The S-layer in M alcaliphilus 20Z consisting of tightly packed, cup-shaped subunits was lost during growth at pH 7.2 (the lowest possible pH) in the absence of NaCl. Hence, osmoadaptation in the methanotrophs studied involves structure/function alterations of cell envelopes and changes in the chemical composition of membranes as well as de novo synthesis of compatible solutes.

Keywords: Key words Methylobacter alcaliphilus 20Z; Methylobacter modestohalophilus 10S; Osmoprotectants; Ectoine; Sucrose; 5-Oxoproline; Phospholipids; Osmoadaptation

Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleavage dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway by John K. Davis; Zhongqi He; Charles C. Somerville; J. C. Spain (pp. 330-339).

Nitrobenzene is degraded to pyruvate and acetaldehyde by Pseudomonas pseudoalcaligenes JS45 via a reductive pathway, and by Comamonas sp. JS765 via an oxidative pathway. Although the initial reactions in the degradation of nitrobenzene by the two bacteria are totally different, the lower pathways are similar and converge at the level of 4-oxalocrotonate. In order to further investigate the biochemical properties and reveal the evolutionary relationships between the two lower pathways, the genes encoding the 2-aminophenol 1,6-dioxygenase were cloned and sequenced. 2-Aminophenol 1,6-dioxygenase from P. pseudoalcaligenes JS45 and catechol 2,3-dioxygenase from Comamonas sp. JS765 were able to act on both catechol and 2-aminophenol, but catechol was a suicide substrate of 2-aminophenol 1,6-dioxygenase. The activity of 2-aminophenol 1,6-dioxygenase was restored after removal of catechol and incubation with ascorbate and FeCl₂. Both the α-subunit (AmnA) and the β-subunit (AmnB) of the dioxygenase from P. pseudoalcaligenes JS45 show a high degree of identity to the corresponding subunits of the ring-fission dioxygenase from Pseudomonas sp. AP-3: 67% for the α-subunit, and 84% for the β-subunit. Sequence similarity studies suggest that the β-subunits of both 2-aminophenol 1,6-dioxygenases are distantly related to homoprotocatechuate 2,3-dioxygenase from Escherichia coli strains W and C and then to catechol 2,3-dioxygenase from Alcaligenes eutrophus. Four active-site-relevant histidines are conserved in AmnB, but not in AmnA. The lack of conserved histidines indicates the absence of an Fe²⁺ binding site in AmnA, which explains the previous observations of only approximately one Fe²⁺ per two subunits in the 2-aminophenol 1,6-dioxygenases from P. pseudoalcaligenes JS45. The 2-aminophenol 1,6-dioxygenase genes are located upstream of the 2-aminomuconic semialdehyde dehydrogenase gene, and a putative member of the YjgF protein family is upstream of the dioxygenase genes. Transcriptional analysis indicates that the YjgF-like protein, 2-aminophenol 1,6-dioxygenase, and 2-aminomuconic semialdehyde dehydrogenase are coordinately transcribed. A putative ORF similar to part of the RNA helicase genes is downstream of the dehydrogenase gene. Both the novel organization of the genes and the phylogeny of the dioxygenases and dehydrogenase indicate that the 2-aminophenol pathway in P. pseudoalcaligenes JS45 represents an example of a distant divergent evolution of meta-cleavage pathways.

Keywords: Key wordsPseudomonas; 2-Aminophenol; Catechol; Dioxygenase; meta-Cleavage pathway; Biodegradation

Sections

Personal tools

Archives of Microbiology (v.172, #5)

Sections

Personal tools

Local resources

Archives of Microbiology (v.172, #5)