The main pigment of the dormant Mycobacterium smegmatis is porphyrin.

Upon transition of Mycobacterium smegmatis into the dormant state, accumulation of a dark brown fluorescent pigment was observed. This pigment gave bright red fluorescence in both cells and the culture medium. Based on 1H-NMR, MALDI and UV spectra, the fluorescent compounds, extracted from the culture medium as well as from the dormant cells, were concluded to be a mixture of free coproporphyrin III and uroporphyrin III and their corresponding methyl esters. A possible significance of porphyrin pigment accumulation in the dormant cells is discussed.

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria.

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) - an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) - most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82-92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes'', well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.

The ability of a recombinant Escherichia coli strain to synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate correlates with its tolerance to in vitro induced oxidative stress and to the bactericidal action of murine peritoneal macrophages.

A number of bacteria are able to synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (BOSS) in response to oxidative stress. Here we show that the ability to synthesize BOSS can be genetically transferred from Corynebacterium ammoniagenes to Escherichia coli. A total DNA library from C. ammoniagenes ATCC 6872 established in the pBluescript SKII+vector backbone was transfected into E. coli XL-1 blue. Recombinant clone 2-31, which was resistant to redox-cycling agents, was selected. NMR studies showed that this clone was able to synthesize BOSS. We also studied the resistance of clone 2-31 to the bactericidal action of macrophages. Clone 2-31 cells had better survival within murine peritoneal macrophages than parental E. coli XL-1-blue cells. Since the ability to synthesize BOSS correlates with increased survival of bacteria within macrophages, we suggest that the pathogenicity of Corynebacteria could be mediated through the synthesis of BOSS.

Lysodektose--a new trisaccharide from Micrococcus lysodeikticus which is transformed into an aminoxyl free radical with the loss of an electron.

A new trisaccharide 6-0-(2-deoxy-2-(N-methyl)-hydroxylamino-beta-D- glucopyranosyl)-alpha,alpha-trehalose named lysodektose has been isolated from Micrococcus lysodeikticus. In oxygenated solutions or in the presence of K3 Fe (CN)6 lysodektose is transformed into a long lived free radical. Spin trapping data are presented and functions are suggested for the substance.

The distribution of electron flow in the branched respiratory chain of Micrococcus luteus.

Endogenous coupled respiration of Micrococcus luteus protoplasts showed a relatively high resistance to low concentrations of KCN, 2-nonyl-4-hydroxyquinoline N-oxide (NQNO) and dicyclohexylcarbodi-imide (DCCD) when the inhibitors were applied individually. In the presence of both KCN and NQNO (or DCCD), O2 uptake was strongly inhibited. The proteolysis of external membrane proteins of protoplasts also induced the high sensitivity of endogenous coupled respiration to low KCN. The effects of NQNO, DCCD and proteolysis were explained by the inhibition of an alternative respiratory system when reducing equivalents passed preferentially down the KCN-sensitive cytochrome oxidase. Uncoupling of the cell membrane increased the electron flow via the cytochrome oxidase-containing respiratory branch. It is suggested that the energy state of cells could control the electron-flow distribution between two branches, and quinones of different levels of reduction could be involved in the mechanism of respiratory branching.

Derivatives of glutamic acid and trehalose which can be transformed into long-living free radicals by the loss of an electron are identified in some bacteria.

A derivative of glutamic acid (ammonigenin) and a trisaccharide named lysodektose which are converted into long-living free radicals by the loss of one electron were isolated from Brevibacterium ammoniagenes and Micrococcus lysodeikticus. Structural formulae suggested for both substances based on ESR-, NMR- and mass spectra, isotopic substitution experiments and other data are: lactone of N-hydroxy-N-(2-carbamoylethyl)-glutamyl-4-amino-2-hydroxybutyric amide and 6-O-[2-deoxy-2-(N-methyl)-hydroxylamino-beta-D-glucopyranosyl]- alpha, alpha-trehalose. Radical forms appear on reversible oxidation of hydroxylamino groups to nitroxyl groups. Participation in the protection of bacterial cells and regulation of their metabolism is suggested for these compounds.

Subunit composition of the H+-ATPase complex from anaerobic bacterium Lactobacillus casei.

The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.

Some peculiarities of functioning of H+-ATPase from the membranes of the anaerobic bacterium Lactobacillus casei.

Radiation inactivation analysis gave the target sizes of 176 +/- 5 kDa and 275 +/- 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1-ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole owing to modification of an amino acid residue(s) with pK greater than 8.5. L. casei ATPase consists of six identical subunits and differs from alpha 3 beta 3 gamma delta epsilon-type F1-ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the 'unisite' conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondrial F1-ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.

Respiratory chain of bacterial membrane: assembly-mobile carriers equilibrium.

The target size of NADH-oxidase activity of M. lysodeikticus isolated membranes for electron radiation is nearly equal to that obtained for NADH-dehydrogenase (about 50 kD). The complete cross-linking of membrane proteins by glutaraldehyde causes an increase of NADH-oxidase target size to 3-3.5 times its original value. Electrons are transported by cross-linked respiratory chain from NADH to O2 with 60-50% effectiveness of that in untreated membranes. It is proposed that electrons are transported through a multi-enzymic complex of individual carriers having limited lifetime with exchange of carriers between different respiratory complexes via lateral diffusion in membrane.