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The zoonotic pathogen Campylobacter jejuni NCTC 11168 uses a complex set of electron transport chains to ensure growth with a variety of electron donors and alternative electron acceptors, some of which are known to be important for host colonization. Many of the key redox proteins essential for electron transfer in this bacterium have N-terminal twin-arginine translocase (TAT) signal sequences that ensure their transport across the cytoplasmic membrane in a folded state. By comparisons of 2D gels of periplasmic extracts, gene fusions and specific enzyme assays in wild-type, tatC mutant and complemented strains, we experimentally verified the TAT dependence of 10 proteins with an N-terminal twin-arginine motif. NrfH, which has a TAT-like motif (LRRKILK), was functional in nitrite reduction in a tatC mutant, and was correctly rejected as a TAT substrate by the TATFIND and TatP prediction programs. However, the hydrogenase subunit HydA is also rejected by TATFIND, but was shown to be TAT-dependent experimentally. The YedY homologue 0j0379 is the only TAT translocated molybdoenzyme of unknown function in C. jejuni; we show that a cj0379c mutant is deficient in chicken colonization and has a nitrosative stress phenotype, suggestive of a possible role for 0j0379 in the reduction of reactive nitrogen species in the periplasm. Only two potential TAT chaperones, NapD and Cj1514, are encoded in the genome. Surprisingly, despite homology to TorD, Oil 514 was shown to be specifically required for the activity of formate dehydrogenase, not trimethylamine N-oxide reductase, and was designated FdhM. DOI 10.1099/mic.0.042788-0

In Gram-negative bacteria, production of adhesion factors and extracellular polysaccharides (EPS) is promoted by the activity of diguanylate cyclases (DGCs), a class of enzymes able to catalyse the synthesis of the signal molecule bis-(3',5')-cyclic di-guanylic acid (c-di-GMP). In this report we show that in Escherichia coli, overexpression of the YddV protein, but not of other DGCs such as AdrA and YcdT, induces the production of the EPS poly-N-acetylglucosamine (PNAG) by stimulating expression of pgaABCD, the PNAG-biosynthetic operon. Stimulation of PNAG production and activation of pgaABCD expression by the YddV protein are abolished by inactivation of its GGDEF motif, responsible for DGC activity. Consistent with the effects of YddV overexpression, inactivation of the yddV gene negatively affects pgaABCD transcription and PNAG-mediated biofilm formation, pgaABCD regulation by the yddV gene also takes place in a mutant carrying a partial deletion of the csrA gene, which encodes the main regulator of pgaABCD expression, suggesting that YddV does not regulate pgaABCD through modulation of CsrA activity. Our results demonstrate that PNAG production does not simply respond to intracellular c-di-GMP concentration, but specifically requires the DGC activity of the YddV protein, thus supporting the notion that in E. coli, c-di-GMP biosynthesis by a given DGC protein triggers regulatory events that lead to activation of specific sets of EPS biosynthetic genes or proteins. DOI 10.1099/mic.0.041350-0

The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional [sup.13]C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39 x [10.sup.-5] [s.sup.-1] and 4.44 x [10.sup.-5] [s.sup.-1]) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to C[O.sub.2]) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.

The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by [sup.1]H-NMR and mass spectroscopy and their antibacterial activity was assayed.

Members of the Chlorobiaceae and Chloroflexaceae are unique among the phototrophic micro-organisms in having a remarkably rich chlorophyll pigment diversity. The physiological regulation of this diversity and its ecological implications are still enigmatic. The bacteriochlorophyll composition of the chlorobiaceae Prosthecochloris aestuarii strain CE 2404 and Chlorobium phaeobacteroides strain UdG 6030 was therefore studied by both HPLC with photodiode array (PDA) detection and liquid chromatography-mass spectrometry (LC-MS). These strains were grown in liquid cultures under green light (480-615 nm) at different light intensities (0.2-55.7 [micro]mol photons [m.sup.-2] [s.sup.-1]), simulating the irradiance regime at different depths of the water column of deep lakes. The specific growth rates of Ptc. aestuarii under green light achieved a maximum of 0.06 [h.sup.-1] at light intensities exceeding 6 [micro]mol photons [m.sup.-2] [s.sup.-1], lower than the maximum observed under white light (approx. 0.1 [h.sup.-1]). The maximal growth rates of Chl. phaeobacteroides under green light were slightly higher (0.07 [h.sup.-1]) than observed for Ptc. aestuarii and were achieved at 3-5 and 4.3 [micro]mol photons [m.sup.-2] [s.sup.-1]. LC-MS/MS analysis of pigment extracts revealed most (> 90 %) BChl c homologues of Ptc. aestuarii to be esterified with farnesol. The homologues differed in mass by multiples of 14 Da, reflecting different alkyl subsituents at positions C-8 and C-12 on the tetrapyrrole macrecycle. The relative proportions of the individual homologues varied only slightly among different light intensities. The specific content of BChl c was maximal at 3-5 [micro]mol photons [m.sup.-2] [s.sup.-1] [400 [+ or -] 150 nmol BChl c (mg [protein).sup.-1]]. In the case of Chl. phaeobacteroides, the specific content of BChl e was maximal at 4.3 [micro]mol photons [m.sup.-2] [s.sup.-1] [115 nmol BChl e (mg [protein).sup.-1]], and this species was characterized by high carotenoid (isorenieratene) contents. The major BChl e forms were esterified with a range of isoprenoid and straight-chain alcohols. The major isoprenoid alcohols comprised mainly farnesol and to a lesser extent geranylgeraniol. The straight-chain alcohols included [C.sub.15], [C.sub.15]: 1, [C.sub.16], [C.sub.16:1] and [C.sub.17]. Interestingly, the proportion of straight alkyl chains over isoprenoid esterified side chains shifted markedly with increasing light intensity: the isoprenoid side chains dominated at low light intensities, while the straight-chain alkyl substituents dominated at higher light intensities. The authors propose that this phenomenon may be explained as a result of changing availability of reducing power, i.e. the highly reduced straight-chain alcohols have a higher biosynthetic demand for [NADPH.sub.2] than the polyunsaturated isoprenoid with the same number of carbon atoms.

The ceplR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyI-L-homoserine lactone (OHL) and N-hexanoyI-L-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-12 (cepl: :[Tp.sup.r]) or K56-R2 (cepR'' Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-12 or K56-R2 compared to the parent strain. Intranasal infections were performed in [Cftr.sup.(-/-)] mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-12 was the least virulent strain and was not invasive in the [Cftr.sup.(-/-)] mice. OHL was readily detected in lung homogenates from [Cftr.sup.(-/-)] mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-12. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1 [beta] and interleukin-6 than those from K56-12-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepl mutant and the parent strain.

Several reports have indicated that the iron-sulfur [Fe-S] assemblly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterised and most highly conserved [Fe-S] assembly proteins are a pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe-S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CplscS and CplscU) and phylogenetic studies (CplscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CplscS and CplscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe-S] assembly.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium Ioti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. Ioti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

Ochratoxin A is an important nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus ochraceus as a polyketide-derived secondary metabolite. A portion of a putative polyketide synthase gene (pks) involved in the biosynthesis of this mycotoxin was cloned by using a suppression subtractive hybridization PCR-based approach. The predicted amino acid sequence of the 1.4 kb clone shared 28-35% identity to acyl transferase regions from fungal polyketide synthases found in the databases. Based on reverse transcription PCR studies, the pks gene is expressed only under ochratoxin A permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted cannot synthesize ochratoxin A. This report is the first of the cloning and characterization of a gene involved in ochratoxin A biosynthesis.

Bacillus subtilis uses glutamine as the best source of nitrogen. In the absence of glutamine, alternative nitrogen sources such as ammonium can be used. Ammonium utilization involves the uptake of the gas or the ammonium ion, the synthesis of glutamine by the glutamine synthetase and the recycling of the glutamate by the glutamate synthase. In this work, ammonium transport in B. subtilis was studied. At high ammonium concentrations, a large fraction of the ammonium is present as ammonia, which may enter the cell via diffusion. In contrast, the ammonium transporter NrgA is required for ammonium utilization at low concentrations or at low pH values when the equilibrium between uncharged ammonia and the ammonium ion is shifted towards ammonium. Moreover, a functional NrgA is essential for the transport of the ammonium analogue methylammonium. NrgA is encoded in the nrgAB operon. The product of the second gene, NrgB, is a member of the PII family of regulatory proteins. In contrast to PII proteins from other organisms, there is no indication for a covalent modification of NrgB in response to the nitrogen supply of the cell. It is demonstrated here that NrgB is localized at the membrane, most likely in association with the ammonium transporter NrgA. The presence of a functional NrgB is required for full-level expression of the nrgAB operon in response to nitrogen limitation, suggesting that NrgB might relay the information on ammonium availability to downstream regulatory factors and thus fine-tune their activity.

The locus lex2, comprising lex2A and lex2B, contributes to the phase-variable expression of lipopolysaccharide (LPS) of Haemophilus influenzae and was found to be present in 74 % of strains investigated, lex2A contains 5'-GCAA repeats which vary in number from 4 to 46 copies between strains. The locus was cloned from the serotype b strains RM7004 and RM153 and showed > 99 % nucleotide sequence identity between these strains and the published lex2 sequence. Disruption of the lex2B gene in strain RM7004 resulted in truncation of some LPS glycoforms, shown by gel fractionation, with only one glycoform reacting with a digalactosidespecific monoclonal antibody, 4C4, compared with four LPS glycoforms in the more elongated LPS of the parent strain. Mass spectrometry and NMR analyses of LPS from the lex2B mutant revealed loss of the terminal digalactoside as well as the second [beta]-glucose extending from the first heptose of the inner core. The authors conclude that Lex2B is the [beta]-(1-4)-glucosyltransferase that adds the second [beta]-glucose to the first [beta]-glucose as part of the oligosaccharide extension from the first heptose of the LPS of strain RM7004. Investigation of the expression of the lex2 locus indicated that the genes are co-transcribed and that both reading frames are required for addition of this second [beta]-glucose in a phase-variable manner.

The cAMP-dependent pathway, which regulates yeast-to-hypha morphogenesis in Candida albicans, is controlled by changes in cAMP levels determined by the processes of synthesis and hydrolysis. Both low- and high-affinity cAMP phosphodiesterases are encoded in the C. albicans genome. CaPDE2, encoding the high-affinity cAMP phosphodiesterase, has been cloned and shown to be toxic in Saccharomyces cerevisiae upon overexpression under pGAL1, but functional under the moderate pMET3, Deletion of CaPDE2 causes elevated cAMP levels and responsiveness to exogenous cAMP, higher sensitivity to heat shook, severe growth defects at 42[degrees]C and highly reduced levels of EFG1 transcription. In vitro in hypha-inducing liquid medium CaPDE2, deletion prohibits normal hyphal, but not pseudohyphal growth. On solid medium capde2 mutants form aberrant hyphae, with fewer branches and almost no lateral buds, which are deficient in hypha-to-yeast reversion. The phenotypic defects of capde2 mutants show that the cAMP-dependent pathway plays specific roles in hyphal and pseudohyphal development, its regulatory role however, being greater in liquid than on solid medium in vitro. The increased expression of CaPDE2 after serum addition correlates well with a drop in cAMP levels following the initial rise in response to the hyphal inducer. These results suggest that Capde2p mediates a desensitization mechanism by lowering basal cAMP levels in response to environmental stimuli in C. albicans.

A two-component regulatory system, cpxR-cpxA, plays an important role in the pH-dependent regulation of virF, a global activator for virulence determinants including invasion genes, in Shigella sonnei. The authors examined whether the cpxR-cpxA homologues have some function in the expression of Salmonella enterica serovar Typhimurium invasion genes via the regulation of hilA, an activator for these genes. In a Salmonella cpxA mutant, the hilA expression level was reduced to less than 10% of that in the parent strain at pH 6.0. This mutant strain also showed undetectable synthesis of an invasion gene product, SipC, at pH 6.0 and reduced cell invasion capacity--as low as 20% of that of the parent. In this mutant, the reduction in hilA expression was much less marked at pH 8.0 than at pH 6.0--no less than 50% of that in the parent, and no significant reduction was observed in either SipC synthesis or cell invasion rate, compared to the parent. Unexpectedly, a Salmonella cpxR mutant strain and the parent showed no apparent difference in all three characteristics described above at either pH. These results indicate that in Salmonella, the sensor kinase CpxA activates hilA, and consequently, invasion genes and cell invasion capacity at pH 6.0. At pH 8.0, however, CpxA does not seem to have a large role in activation of these factors. Further, the results show that this CpxA-mediated activation does not require its putative cognate response regulator, CpxR. This suggests that CpxA may interact with regulator(s) other than CpxR to achieve activation at low pH.

Members of the Mycobacterium avium complex are distinguished by the presence of highly antigenic surface molecules called glycopeptidolipids (GPLs) and the oligosaccharide portion of the serovar-specific GPL defines the 28 serovars. Previously, the genomic region (ser2) encoding the enzymes responsible for the glycosylation of the lipopeptide core to generate the serovar-2-specific GPLs has been described. In this work, the ser2 gene clusters of M. avium serovar 2 strains 2151 and TMC 724 were fully sequenced and compared to the homologous regions of M. avium serovar 1 strain 104, M. avium subsp, paratuberculosis and M. avium subsp. silvaticum. It was also determined that 104Rg, a mutant of strain 104 that produces truncated GPLs, lost several GPL biosynthesis genes by deletion. This comparison, together with analysis of protein similarities, supports a biosynthetic model in which serovar-2-specific GPLs are synthesized from a serovar-1-specific GPL intermediate that is derived from a non-specific GPL precursor. We also identified a gene encoding an enzyme that is necessary for the biosynthesis of serovar-3-and 9-specific GPLs, but not serovar-2-specific GPLs, suggesting that the different serovars may have evolved from the acquisition or loss of genetic information, in addition, a subcluster of genes for the biosynthesis and transfer of fucose, which are needed to make serovar-specific GPLs such as those of serovar 2, is found in the non-GPL-producing M. avium subspecies paratuberculosis and silvaticum.

The melanogenic marine bacterium Marinomonas mediterranea synthesizes R-bodies as revealed by transmission electron microscopy. These structures were previously described in some obligate symbionts of paramecia and some free-living bacteria, none of which was isolated from sea water. In other micro-organisms, the synthesis of R-bodies has been related to extrachromosomal elements. Accordingly, M. mediterranea induction by mitomycin C or UV radiation resulted in the production of defective phages resembling bacteriocins, indicating that it is a lysogenic bacterium. Two mitomycin-C-resistant strains defective in prophage replication have been isolated. These mutants, and the previously obtained strains ngC1, T102 and T103, the latter mutated in the ppoS gene encoding a sensor histidine kinase, are affected not only in phage replication but also in polyphenol oxidase activities and melanin synthesis, suggesting a relationship between the control of all these processes.

A remarkable cell shape change was observed in Bacillus subtilis strain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KN[O.sub.3] was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0*1% KN[O.sub.3] and the shape-changed cells grown without KN[O.sub.3] revealed that the expression of the branched-chain [alpha]-keto acid dehydrogenase complex (coded by the bfmB gene locus) was elevated in the shape-changed cells. Inactivation of the bfmB locus resulted in the repression of cell shape change, and cells in which bfmB expression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0*1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.

Mid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1-MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat. To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes. These include WSC1 and ROM2, upstream elements in cell integrity pathway signalling, and FKS1 and SMI1, required for 1,3-[beta]-glucan synthesis. These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way. To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZE01 (YOL109w), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane. Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway. Consistent with this, zeo1-deficient cells suppress the growth defect of mutants in the Rho1p GDP-GTP exchange factor Rom2p, while exacerbating the growth defect of sac7[DELTA] mutants at 37[degrees]C. In contrast, mid2[DELTA] mutants have opposing effects to zeo1[DELTA] mutants, being synthetically lethal with rom2[DELTA], and suppressing an 18[degrees]C growth defect of sac7[DELTA], while overexpression of MID2 rescues a rom2[DELTA] 37[degrees]C growth defect. Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1-MPK1 cell integrity pathway.

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58*0 kDa. The enzyme had an isoelectric point of around pH 6*9. The optimum pH for enzyme activity was around 3*0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40[degrees]C and stable up to 50[degrees]C. The enzyme contained 8*6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. [beta](3,4-Dihydroxyphenyl)alanine (L-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of L-DOPA was identified as L-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

Pseudomonas aeruginosa produces extracellular glycolipids composed of L-rhamnose and 3-hydroxyalkanoic acid called rhamnolipids. Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood. Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by the rhlAB operon, which catalyses the transfer of TDP-L-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths. RhlB is the catalytic protein of the rhamnosyltransferase, rhlA is indispensable for rhamnolipid synthesis, but its function is unknown. Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs by P. aeruginosa was detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis. In this report, evidence is presented indicating that rhlA is required for production of HAAs and that these HAAs display potent surface-active properties. P. aeruginosa can colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents. Using rhlA and rhlB mutants it was observed that swarming requires the expression of the rhlA gene but does not necessitate rhamnolipid production, as HAAs act as surfactants. Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decrease rhlA expression and swarming motility.

Cryptococcus neoformans is a pathogenic fungus that produces melanin when incubated in the presence of certain phenolic substrates such as L-3, 4-dihydroxyphenylalanine (L-dopa). Melanin is an enigmatic polymer that is deposited in the cell wall and contributes to virulence. Substantial progress has been made in understanding the synthesis of melanin and the mechanisms by which it contributes to virulence, but relatively little is known about how melanin is rearranged during growth and budding. In this study we used transmission and scanning electron microscopy and immunofluorescence of melanized cells and melanin 'ghosts' to study the process of melanization during replication. Budding in melanized C. neoformans results in focal disruption of cell-wall melanin at the bud site. In the presence of L-dopa, bud-related melanin defects are repaired and daughter cells are melanized. However, in the absence of substrate, mother cells cannot repair their melanin defects and daughter cells are non-melanized. Hence, melanin in the parent cell is not carried to the daughter cells, but rather is synthesized de novo in buds. These results imply that melanin remodelling occurs during cell growth in a process that involves degradation and synthesis at sites of budding.

This paper extends, with computer techniques, the authors' previous work on the kinetics of pole wall and sidewall synthesis in Escherichia coil These findings extend the conclusion that the nascent poles are made of entirely new material and that no new material is inserted into old poles. This requires re-evaluation of ideas in the literature about wall growth and cell division. Mechanisms of various types have been suggested for the growth of Gram-negative rod-shaped bacteria and these will also require major re-evaluation because of the finding, reported here, that the sidewall is made in several modes: patches of new murein, bands of new material largely going circumferentially around the cell, and areas of the sidewall that are enlarged by an intimate and regular admixture of new with the old muropeptides.

Neisseria meningitidis is a Gram-negative bacterium which is an important causative agent of septicaemia and meningitis. LuxS has been shown to be involved in the biosynthesis of a quorum sensing molecule, autoinducer-2 (AI-2), known to play a role in virulence in Escherichia coli, as well as other bacteria. Evidence that serogroup B of N. meningitidis produces AI-2, along with the observation that a luxS mutant of this strain had attenuated virulence in an infant rat model of bacteraemia, led to further investigation of the role of this quorum sensing molecule in N. meningitidis. In this study, it is demonstrated that AI-2 is not involved in regulating growth of meningococci, either in culture or in contact with epithelial cells. Furthermore, transcriptional profiling using DNA microarrays shows an absence of the concerted regulation seen in other bacteria. Taken together, these data suggest that in N. meningitidis, AI-2 may be a metabolic by-product and not a cell-to-cell signalling molecule.

The pleiotropic phenotype of an auxotrophic purL mutant (SVQ295) of Sinorhizobium fredii HH103 has been investigated. SVQ295 forms colonies that are translucent, produce more slime and absorb less Congo red than those of wild-type strain HH103. SVQ295 did not grow in minimal medium unless the culture was supplemented with thiamin and adenine or with thiamin and AICA-riboside (5-aminoimidazole-4-carboxamide 1-[beta]-D-ribofuranoside), an intermediate of purine biosynthesis. Bacterial cultures supplemented with AICA-riboside or adenine reached the same culture density, although the doubling time of SVQ295 cultures containing AICA-riboside was clearly longer. S. fredii SVQ295 induced pseudonodules on Glycine max and failed to nodulate six different legumes. On Glycyrrhiza uralensis, however, nodules showing nitrogenase activity and containing infected plant cells were formed. SVQ295 showed auto-agglutination when grown in liquid TY medium and its lipopolysaccharide (LPS) electrophoretic profile differed from that of its parental strain HH103-1. In addition, four monoclonal antibodies that recognize the LPS of S. fredii HH103 failed to recognize the LPS produced by SVQ295. In contrast, [sup.1]H-NMR spectra of K-antigen capsular polysaccharides (KPS) produced by SVQ295 and the wild-type strain HH 103 were similar. Co-inoculation of soybean plants with SVQ295 and SVQ116 (a nodA mutant derivative of HH103) produced nitrogen-fixing nodules that were only occupied by SVQ116.

The respiratory inhibitor cyanide stimulates growth of the ethanologenic bacterium Zymomonas mobilis, perhaps by diverting reducing equivalents from respiration to ethanol synthesis, thereby minimizing accumulation of toxic acetaldehyde. This study sought to identify cyanide-sensitive components of respiration. In aerobically grown, permeabilized Z. mobilis cells, addition of 200 [micro]M cyanide caused gradual inhibition of ADH II, the iron-containing alcohol dehydrogenase isoenzyme, which, in aerobic cultures, might be oxidizing ethanol and supplying NADH to the respiratory chain. In membrane preparations, NADH oxidase was inhibited more rapidly, but to a lesser extent, than ADH II. The time-course of inhibition of whole-cell respiration resembled that of NADH oxidase, yet the inhibition was almost complete, and was accompanied by an increase of intracellular NADH concentration. Cyanide did not significantly affect the activity of ADH I, the zinc-containing alcohol dehydrogenase isoenzyme. When an aerobic batch culture was grown in the presence of 200 [micro]M cyanide, cyanide-resistant ADH II activity was observed, its appearance correlating with the onset of respiration. It is concluded that the membrane-associated respiratory chain, but not ADH II, is responsible for the whole-cell cyanide sensitivity, while the cyanide-resistant ADH II is needed for respiration in the presence of cyanide, and represents an adaptive response of Z. mobilis to cyanide, analogous to the induction of alternative terminal oxidases in other bacteria.

The analysis of the available Corynebacterium genome sequence data led to the proposal of the presence of all three known pathways for trehalose biosynthesis in bacteria, i.e. trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway), from malto-oligosaccharides or [alpha]-1,4-glucans (TreY-TreZ pathway), or from maltose (TreS pathway). Inactivation of only one of the three pathways by chromosomal deletion did not have a severe impact on C. glutamicum growth, while the simultaneous inactivation of the OtsA-OtsB and TreY-TreZ pathway or of all three pathways resulted in the inability of the corresponding mutants to synthesize trehalose and to grow efficiently on various sugar substrates in minimal media. This growth defect was largely reversed by the addition of trehalose to the culture broth. In addition, a possible pathway for glycogen synthesis from ADP-glucose involving glycogen synthase (GlgA) was discovered. C. glutamicum was found to accumulate significant amounts of glycogen when grown under conditions of sugar excess. Insertional inactivation of the chromosomal glgA gene led to the failure of C. glutamicum cells to accumulate glycogen and to the abolition of trehalose production in a [DELTA]otsAB background, demonstrating that trehalose production via the TreY-TreZ pathway is dependent on a functional glycogen biosynthetic route. The trehalose-non-producing mutant with inactivated OtsA-OtsB and TreY-TreZ pathways displayed an altered cell wall lipid composition when grown in minimal broth in the absence of trehalose. Under these conditions, the mutant lacked both major trehalose-containing glycolipids, i.e. trehalose monocorynomycolate and trehalose dicorynomycolate, in its cell wall lipid fraction. The results suggest that a dramatically altered cell wall lipid bilayer of trehalose-less C. glutamicum mutants may be responsible for the observed growth deficiency of such strains in minimal medium. The results of the genetic and physiological dissection of trehalose biosynthesis in C. glutamicum reported here may be of general relevance for the whole phylogenetic group of mycolic-acid-containing coryneform bacteria.

Medermycin is a Streptomyces aromatic C-glycoside antibiotic classified in the benzoisochromanequinones (BIQs), which presents several interesting biosynthetic problems concerning polyketide synthase (PKS), post-PKS tailoring and deoxysugar pathways. The biosynthetic gene cluster for medermycin (the med cluster) was cloned from Streptomyces sp. AM-7161. Completeness of the clone was proved by the heterologous expression of a cosmid carrying the entire med cluster in Streptomyces coelicolor CH999 to produce medermycin. The DNA sequence of the cosmid (36 202 bp) revealed 34 complete ORFs, with an incomplete ORF at either end. Functional assignment of the deduced products was made for PKS and biosynthetically related enzymes, tailoring steps including strereochemical control, oxidation, angolosamine pathway, C-glycosylation, and regulation. The med cluster was estimated to be about 30 kb long, covering 29 ORFs. An unusual characteristic of the cluster is the disconnected organization of the minimal PKS genes: med-ORF23 encoding the acyl carrier protein is 20 kb apart from med-ORF1 and med-ORF2 for the two ketosynthase components. Secondly, the six genes (med-ORF14, 15, 16, 17, 18 and 20) for the biosynthesis of the deoxysugar, angolosamine, are all contiguous. Finally, the finding of a glycosyltransferase gene, med-ORF8, suggests a possible involvement of conventional C-glycosylation in medermycin biosynthesis. Comparison among the three complete BIQ gene clusters--med and those for actinorhodin (act) and granaticin (gra)--revealed some common genes whose deduced functions are unavailable from database searches (the 'unknowns'). An example is med-ORF5, a homologue of actVI-ORF3 and gra-ORF18, which was highlighted by a recent proteomic analysis of S. coelicolor A3(2).

Research has been conducted on Mycobacterium tuberculosis strains with defects in amino acid biosynthesis pathway. Results indicate that the gene which encodes shikimate kinase catalyzing chorismate biosynthesis steps is essential in M. tuberculosis.

Research has been conducted on the sulfur-containing polyhydroxyalkanoates consisting of hydroxypropylthioalkanoic acid which contains thioether groups in the side chains. The biosynthesis of these polyhydroxyalkanoates has been carried out and the details are presented.

Research has been conducted on the fatty acid elongation system FAS-II involved in the mycolic acid biosynthesis. The enzymological and structural analyses of protein MabA, a potential component of FAS-II, have been carried out and the results indicate that the specific structural features of this protein will allow the new antibiotic development.

Research has been conducted on the Caulobacter crescentus. The ability of C. crescentus to produce polyhydroxybutyrate in the presence of the excess carbon source has been investigated and the results are reported.

Research has been conducted on the Streptomyces venezuelae ISP5230. The cloning of the chloramphenicol biosynthesis cluster directed to the regions of the S. venezuelae ISP5230 chromosome flanking pabAB has been carried out and investigated.

Research has been conducted on the catecholate siderophore biosynthesis. The isolation of the gene required for this biosynthesis has been carried out.

Research describes derivation and analysis of single and double mutants of Kluyveromyces lactis impaired or lacking in glutamate synthase (GOGAT) and GOGAT and NADP-glutamate dehydrogenase pathway respectively. Results demonstrate that glutamate biosynthesis in this bacterium requires combined action of both.

The transfer of the plant pathogenic bacteria Erwinia amylovora ams genes and E. stewartii cps genes into the mutants of other species that are unable to produce exopolysaccharides (EPS) stimulates the production of stewartan and amylovoran, respectively. However, the E. stewartii cpsK mutant complemented with ams genes produces stewartan. Both the EPSs are acylated in E. amylovora but not in E. stewartii. The synthesis of amylovoran in E. stewartii restores its virulence, but stewartan is unable to do so in E. amylovora.

A study of the construction of nine 'mob' mutants of Escherichia coli by gamma-placMu9 mutagenesis in which the mob promoter controls the activity of beta-galactosidase shows that the mob is expressed at very low levels under anaerobic and aerobic growth conditions. The mob locus of Escherichia coli facilitates the synthesis of molybdoprotein guanine dinucleotide, a molybdenum cofactor from GTP and molybdopterin. The mob locus DNA sequence show that the DNA encodes two ORF's present as a single transcription unit with polypeptide products of 21642 Da and 19362 Da.

Aflatoxins are biologically active secondary metabolites made by Aspergillus parasiticus, A. nomius and A. flavus fungi and can be deadly to humans if they are ingested. Genes involved in aflatoxin biosynthesis can be cloned to help generate understanding of the molecular biology that creates the toxins, as the cloned genes extract the molecular mechanisms that controls the expression and the timing of these genes. Analysis of the biosynthesis can provide insight on how to prevent the toxins from forming in foods.

Several enzymes catalyze the biosynthesis of tentoxin by Alternaria alternata, which occurs through a thiotemplate mechanism, similar to that in various cyclic peptides. Sections of mycelial mats incubated in buffer aid this physiological study. The growth of tentoxin is rapid between 9 and 12 days from the start of the synthesis. There is an enhancement in the rate of tentoxin release from the cells at pH values more or less than 7. This synthesis is aided by precursor amino acids.

Physiological, cellular and molecular investigations on hyphal growth of fungi reveal that the microvesicles having membranes and enzymes which expand the apex are involved in the growth of hyphae. The microvesicles are found in the tapering apical region where cell extension and cell wall biosynthesis occur. The enzymes and membranes in the microvesicles are translocated through the hypha to cell surface at the apical region through microtubules. At the apical region of the hypha these enzymes and membranes act on cells resulting in the growth of the hyphae.

The mutant gene (pro BA) responsible for overproduction of proline in Serratia marsescens was sequenced and characterized. Sequence comparisons showed that the mutant contained two mutations, a G-C to A-T transversion at nucleotide 127 (Ppro 2) within the promoter region and a G-C to A-T transversion at nucleotide 531 (pro B1), which caused an alanine to valine substitution.. The pro B1 mutation caused an insensitivity to feedback inhibition by proline, while the Ppro 2 mutation caused an up-mutation in the promoter.

Research has been conducted on pSV1 plasmid from Streptomyces violaceoruber SANK95570 which carries methylenomycin biosynthetic gene cluster. The authors suggest the possibility that this gene cluster has been transferred horizontally in the evolutionary past.

Research has been conducted on mshB gene, the key enzyme in mycothiol biosynthesis. Results demonstrate that the disruption of this gene in Mycobacterium smegmatis has led to mycothiol production decrease.

Research has been conducted on Acinetobacter baumannii 8399 clinical isolates which secrete dihydroxybenzoic acid and catechol siderophore. The authors describe the identification and characterization of genes coding for putative siderophore transport proteins.

Research has been conducted on gene encoding Leuconostoc mesenteroides Lcc4 dextransucrase DsrD. The authors describe the isolation of this gene via the use of PCR, and demonstrate that dextransucrase can be expressed and secreted in a heterologous host and drive dextrin synthesis.

Research has been conducted on opportunistic respiratory pathogen Burkholderia cepacia. The authors describe the cloning and sequencing genes from a pilus gene cluster, and the results demonstrate that gene products are sufficient for pilus assembly on bacterial surface.

Research has been conducted on CobT enzyme of Salmonella typhimurium. The authors demonstrate that this enzyme has NAD (super)+ -dependent ADPribosyltransferase activity.

Research has been conducted on fluorescent pseudomonas which sectrete pyoverdines. The authors report the identification of genes required for pyoverdine synthesis.

Research has been conducted on curli and cellulose synthesis regulator, CsgD. Results suggest that CsgD upregulates glyA in facilitating curli synthesis.

Research has been conducted on pneumoccoccal serotypes 8 and 37 which have been generated in sorbarods. The authors describe the capsule phase variations of these serotypes and demonstrate that the molecular mechanisms of phase variations are similar to those found in serotype 3 pneumoccocci.

Research has been conducted on exopolysaccharide methanolan. The synthesis of this methanolan has been investigated via the use of methylotroph Methylobacillus sp. strain 12S, and the results are reported.