Sudhamoy Ghosh

Iron Transport in Azospirillum brasilense: Role of the Siderophore Spirilobactin

SUMMARY: A catechol-type siderophore was secreted by Azospirillum brasilense in the growth medium when the cells became iron-deficient. The siderophore, which we call spirilobactin, was purified and a partial characterization of its structure by hydrolysis revealed that it contained 2,3-dihydroxybenzoic acid, ornithine and serine in equimolar amounts. A high-affinity iron transport system capable of 59Fe uptake from a 59Fe(III)-spirilobactin complex was also induced in A. brasilense grown under iron deficiency. Kinetic studies on the iron uptake system revealed that it was carrier-mediated, with K m and V max values of 0·23 μm and 0·27 nmol Fe min−1(mg cell protein)−1, respectively. Dependence of iron uptake on an energized membrane, its insensitivity to arsenate and inhibition by protonophores suggest that transport is driven by the proton gradient across the membrane. Iron-deficient Azospirillum lipoferum transported 59Fe from the 59Fe(III)-spirilobactin complex (at a rate 3-fold lower than that of A. brasilense) but A. amazonense failed to show uptake of iron under the same conditions.

Isolation and Characterization of the Outer Membrane Proteins of Azospirillum brasilense

SUMMARY: The outer membrane of Azospirillum brasilense was isolated from the total membrane fraction by sucrose density gradient centrifugation and by Sarkosyl extraction; both preparations showed an identical outer membrane protein profile in slab gels after electrophoresis under denaturing conditions. The profile showed a major 42 kDa protein constituting about 60% of the total outer membrane proteins. This major protein and a minor 40 kDa protein were tightly but non-covalently associated with peptidoglycan. In addition, a 23 kDa heat-modifiable protein, resistant to trypsin digestion, was detected in the outer membrane. Growth of A. brasilense under iron deficiency induced four additional high molecular mass proteins (87, 83, 78 and 72 kDa) in the outer membrane. A comparison of the outer membrane protein profiles of the three different Azospirillum species suggests that such profiles might be useful in taxonomic classification.

Synthesis of diene prostaglandins in freshwater fish.

Biosynthesis of PGD2, PGE2 and PGF2α in four species of freshwter fish,Tilapia mossambica, Cyprinus carpio, Heteropneustes fossilis andClarius batrichus was studied. Both arachidonic acid and PGH2 were used as substrates. When PGH2 replaced arachidonic acid in the enzymic reaction, there was a 3- to 4-fold increase in PGE2 synthesis, but no such increase in the synthesis of PGF2α and PGD2 was observed.

Preferential oxidation of linolenic acid compared to linoleic acid in the liver of catfish (Heteropneustes fossilis and Clarias batrachus)

The fate of [1-14C] linoleic acid and [1-14C] linolenic acid in the liver slices and also in the liver tissues of live carnivorous catfish,Heteropneustes fossilis andClarias batrachus, was studied. Incorporation of the fatty acids into different lipid classes in the live fish differed greatly from the tissue slices, indicating certain physiological control operative in vivo. The extent of desaturation and chain elongation of linoleic and linolenic acids into long-chain polyunsaturated fatty acids was low. Linolenic acid was oxidized (thus labeling the saturated fatty acid with liberated14C-acetyl-CoA) in preference to linoleic acid, and this oxidation also seemed to be under physiological control since both of the fatty acids were poorly oxidized in the tissue slices and in the killed fish. These fish can therefore recognize the difference in the acyl chain structures of linoleate and linolenate. The higher oxidation of liolenic acid and poor capacity for its conversion to longer chain, highly unsaturated derivatives indicates a higher demand for the dietary supply of these essential fatty acids in these two species.

Identification of a heat-stable palmitoyl/oleoyl specific acyl-acyl carrier protein thioesterase in developing seeds of Madhuca butyracea.

Seed fat formation in Madhuca butyracea (also known as Bassia butyracea), a sub-Himalayan tree of Sapotaceae family, was investigated during the seed development period from 45 DAF (days after flowering) to near maturity at 93 DAF. Although the lipid content per seed kernel increased from 2.5 mg (1.85%, w/w) at 45 DAF to 525 mg (37.5%, w/w) at 93 DAF, the composition of major fatty acids in the lipid did not change appreciably from the early (palmiate, 54%; oleate, 22%; stearate, 2.5% and linoleate, 13%) to the mature stage (palmitate, 66%; oleate, 27.5%; stearate, 2.5% and linoleate, 3%), indicating high content of palmitate in its seed fat (local name Phulwara fat). To investigate the possible role of any palmitoyl specific fatty acyl-ACP thioesterase (FAT) that could be responsible for the accumulation of this unique high-palmitate fat in its seed, FAT activities in the extracts of developing seed kernels (as well as that of young leaves and flower buds) were analyzed. In addition to housekeeping oleoyl preferring LC-FAT (apparent pI, 8.2), a novel FAT showing almost equal substrate specificity for palmitoyl-and oleoyl-ACP, which we termed as PO-FAT, was found at high levels in seed kernels and to a smaller extent in leaves and flower buds. The enzyme (PO-FAT) was purified and biochemically characterized. The relative rate of substrate (at 2 μM) hydrolysis by the purified enzyme was 100:90:30:7 for palmitoyl-, oleoyl-, stearoyl- and lauroyl-ACP, respectively, and the Vmax/Km ratios for the first three substrates were 100: 73: 47, respectively. The enzyme was found to be a heat-stable basic protein (apparent pI, 9.7) showing its optimum activity at pH 9.0.It is highly probable that the enzyme PO-FAT plays a pivotal role in determining the fatty acid composition of the palmitate-rich Phulwara fat in developing seeds of the plant.

Acyl carrier protein of Azospirillum brasilense : properties of the purified protein and sequencing of the corresponding gene, acpP

Acyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in Escherichia coli is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene (acpP) from Azospirillum brasilense using conventional methods. Therefore, ACP from A. brasilense was purified to homogeneity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the acpP region generated by two oppositely oriented internal primers designed from the partial acpP gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2.0-3.0 x 10(4) molecules per cell), (b) compared to E. coli ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.

Deposition of stearate-oleate rich seed fat in Mangifera indica is mediated by a FatA type acyl-ACP thioesterase.

Although the mechanism of accumulation of C8-C16 saturated fatty acids in seed oils has been well-studied, the control of stearic (C18:0) acid deposition in high stearate seed fat is still unclear. We investigated the mechanism that regulates high level of stearate and oleate (C18:1) accumulation in mango (Mangifera indica) seeds during its development, and examined the seed plastid extracts for induction of any specialized fatty acyl-ACP thioesterase (Fat) that may control this high level of deposition. Though the specificity of the Fat enzymes does not account directly for the fatty acid composition of mango seeds, our result suggested that an induced synthesis of a FatA type of thioesterase could be responsible for the high content of oleate and stearate in its seed fat. The major thioesterase from developing seed kernel was purified to near homogeneity, and characterized as a heat-labile, dimeric, neutral protein with relative substrate specificity of 100:35:1.8 towards oleoyl-, stearoyl- and palmitoyl-ACP, respectively. This enzyme was confirmed as Mi FatA by mass spectrometric analysis. Additionally, a heat-stable FatB type enzyme (Mi FatB) was also partially purified, with relative substrate specificity for the same substrates as 9:8.5:100, respectively. Mi FatA is an enzyme of great biotechnological interest because of its involvement in the regulation of stearate rich seed fat in mango.

Effect of dimethylsulfoxide on derepression of nitrogenase in Spirillum lipoferum

Spirillum lipoferum can fix Nz in association with the roots of maize and certain tropical grasses, and partially relieve the dependence of these plants for growth on fixed N [ 11. The potentiality of this bacterium, as an agent of associative symbiosis, depends however on its ability to export NH: outside the cell under the conditions of Naase derepression. In general, Naase of Na fixing bacteria are highly susceptible to ammonia repression [2,3], and N,ase biosynthesis occurs only when a deficiency of ammonia exists within the cells making it impossible for them to export ammonia synthesized from N,. On the other hand, the nif genes of the most useful symbiotic bacteria belonging to Rhizobium spp., when present in the bacteroid form in root-nodules, appear to remain in a derepressed state even in an environment of free ammonia which they export to the cytoplasm of the host cells [3]. It is not clear how ammonia exerts its powerful repression effect on nif in most nitrogen fixing bacteria, and why this repression is particularly weak in the bacteroids of Rhizobium spp. The repression of nif genes by NG can however be overcome in Klebsiella pneumoniae by certain mutations in gin or gin regulatory genes [4,5], and in most other Nz-fixing organisms by adding MSX [3,6,7], a structural analogue of glutamine that strongly inhibits GS [8]. As interpretations of these results have been controversial [3,5,9], we looked for an alternative method of con-

Identification of a new deoxyendonuclease from Escherichia coli that preferentially cleaves supercoiled plasmid DNA

not received Deoxyendonuclease Supercoiled DNA Periplasmic enzyme Endonuclease, of E. coli

Lack of expression of RP4-specified β-lactamase in Azospirillum brasilense

Abstract Plasmid RP4, which normally confers resistance to ampicillin (Apr), tetracycline (Tcr), and kanamycin (Kmr) to its hosts, failed to express enhanced Apr when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic β-lactamase. Even in a β-lactamase-deficient mutant, A. brasilense RG-D16, no increase in β-lactamase or significant Apr appeared following transfer of RP4. However, A. brasilense RG (RP4) and A. brasilense RG-D16 (RP4) did exhibit Tcr Kmr. When RP4 was transferred back from A. brasilense to E. coli all three drug resistances and β-lactamase activity were fully expressed.