Regulation of the <Emphasis Type="Italic">ALBINO3</Emphasis>-mediated transition to flowering in <Emphasis Type="Italic">Arabidopsis</Emphasis> depends on the expression of <Emphasis Type="Italic">CO</Emphasis> and <Emphasis Type="Italic">GA1</Emphasis>

ALBINO3, a homologue of PPF1 in Arabidopsis, encodes a chloroplast protein, and is essential for chloroplast differentiation. In the present study, ALBINO3(−) transgenic plants exhibited a significant decrease in both the number of rosette leaves at bolting and the days before bolting, suggesting the important roles of ALBINO3 in regulating flowering during non-inductive short-day photoperiods. ALBINO3 mRNA was apparently accumulated in shoot apical meristem and floral meristems around the shoot apical meristem in wild-type plants. ALBINO3 might be predominantly involved in inducing the floral repression pathway by activating the expression of TFL1, and by suppressing the expression of LFY, respectively, in the shoot apical meristem. Moreover, the function of ALBINO3 in regulating flowering transition depended on the expression of CO and GA1, because ALBINO3 might function in the downstream integration of the photoperiod-dependent and the photoperiod-independent pathways. These results suggest that ALBINO3 may have an important integrative function in the flowering process in Arabidopsis.     

Structure and expression of gene <Emphasis Type="Italic">vfr</Emphasis> in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Gene vfr of Pseudomonas chlororaphis 449 previously described only in Pseudomonas aeruginosa was identified, cloned, and sequenced; its localization in the chromosome was determined. Amino acid sequence of the protein encoded by gene vfr in P. chlororaphis 449 was shown to have a 83% identity with the Vfr protein of P. aeruginosa PAO1 and a 63% identity with the CRP protein of Escherichia coli. Amino acid residues that ensure the most important structural properties of the CRP protein, i.e., its binding to cAMP, RNA polymerase, and DNA, were identical or highly conserved in Vfr proteins of P. aeruginosa and P. chlororaphis 449. The cloned vfr gene of P. chlororaphis 449 was complemented partially the mutation at gene crp in cells of E. coli AM306 enhancing ten times synthesis of β-galactosidase dependent on the CRP protein. Unlike P. aeruginosa, the Vfr protein in cells of P. chlororaphis 449 does not participate in the regulation of synthesis of N-acyl-homoserine lactones.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Cystic Fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro.     

Phosphorus cycling between the colonial cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and attached bacteria, <Emphasis Type="Italic">Pseudomonas</Emphasis>

Phosphorus (P) transfer between Microcystis aeruginosa and the attached bacterium Pseudomonas was studied using radioactive P (³²P) and green fluorescence protein-labeled Pseudomonas. M. aeruginosa in P-starved condition took up most ³²P (70%) in water and about 50% of ³²P in ³²P-saturated bacteria in individual experiments. However, only 26% of ³²P in the ³²P-saturated M. aeruginosa was transferred to P-starved bacteria. The P-starved M. aeruginosa had an advantage to take up P over the bacteria and its growth rates and abundance were higher in combined cultures, with bacteria as the biotic P source. The rate of P transfer from bacteria to the cyanobacteria was slow. P cycles predominantly between M. aeruginosa and Pseudomonas with little variation in the water. This ability is very useful for the colony-forming M. aeruginosa, especially if phosphate concentrations in water are low during water bloom periods.     

Effects of <Emphasis Type="Italic">dapA</Emphasis> gene deletion on coronatine biosynthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">glycinea</Emphasis> PG4180

Coronatine (COR) is a structural and functional analogue of jasmonic acid that might be employed in agriculture to elicit plant resistance against various aggressors. However, the yield of COR is low both in chemosynthesis and biosynthesis, so broad investigation of COR is difficult. Coronatine combines two distinct components: coronafacic acid (CFA) and coronamic acid (CMA). Synthesis of both CMA and CFA is involved in l-isoleucine metabolism, so the objective of this work was to investigate if COR production can be improved by regulating amino acid biosynthesis in P. syringae pv. glycinea. Inhibition of dihydrodipicolinate synthase was achieved by removing the dapA gene via homologous recombination, which resulted in a COR yield by the dapA ⁻ mutant of about 1.5-fold greater than the wild strain. Thus, regulation of amino acid metabolism is a feasible way to increase COR production, which could be a more effective method than adding substrates into culture medium.     

<Emphasis Type="Italic">Rab11</Emphasis> is required for myoblast fusion in <Emphasis Type="Italic">Drosophila</Emphasis>

Rab11, an evolutionarily conserved, ubiquitously expressed subfamily of small monomeric Rab GTPases, has been implicated in regulating vesicular trafficking through the recycling of endosomal compartment. In order to gain an insight into the role of this gene in myogenesis during embryonic development, we have studied the expression pattern of Rab11 in mesoderm during muscle differentiation in Drosophila embryo. When dominant-negative or constitutively active Drosophila Rab11 proteins are expressed or Rab11 is reduced via double-stranded RNA in muscle precursors, they cause partial failure of myoblast fusion and show anomalies in the shape of the muscle fibres. Our results suggest that Rab11 plays no role in cell fate specification in muscle precursors but is required late in the process of myoblast fusion. This work was supported by grants from the DST (to J.K.R.) and SRF from ICMR, New Delhi (to T.B.).     

Rhamnolipid-producing thermophilic bacteria of species <Emphasis Type="Italic">Thermus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis>

Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans.     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.     

A three-phase <Emphasis Type="Italic">in-vitro</Emphasis> system for studying <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> adhesion and biofilm formation upon hydrogel contact lenses

Background  

Pseudomonas aeruginosa is commonly associated with contact lens (CL) -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented.     

Alginate production and <Emphasis Type="Italic">alg8</Emphasis> gene expression by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> in continuous cultures

Alginates are polysaccharides that are used as thickening agents, stabilizers, and emulsifiers in various industries. These biopolymers are produced by fermentation with a limited understanding of the processes occurring at the cellular level. The objective of this study was to evaluate the effects of agitation rate and inlet sucrose concentrations (ISC) on alginate production and the expression of the genes encoding for alginate-lyases (algL) and the catalytic subunit of the alginate polymerase complex (alg8) in chemostat cultures of Azotobacter vinelandii ATCC 9046. Increased alginate production (2.4 g l⁻¹) and a higher specific alginate production rate (0.1 g g⁻¹ h⁻¹) were obtained at an ISC of 15 g l⁻¹. Carbon recovery of about 100% was obtained at an ISC of 10 g l⁻¹, whereas it was close to 50% at higher ISCs, suggesting that cells growing at lower sucrose feed rates utilize the carbon source more efficiently. In each of the steady states evaluated, an increase in algL gene expression was not related to a decrease in alginate molecular weight, whereas an increase in the molecular weight of alginate was linked to higher alg8 gene expression, demonstrating a relationship between the alg8 gene and alginate polymerization in A. vinelandii for the first time. The results obtained provide a possible explanation for changes observed in the molecular weight of alginate synthesized and this knowledge can be used to build a recombinant strain able to overexpress alg8 in order to produce alginates with higher molecular weights.     

Expression and characterization of (<Emphasis Type="Italic">R</Emphasis>)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4 _Pa from Pseudomonas aeruginosa, termed phaJ4 _Pp, was identified. The gene products of phaJ1 _Pp, which was identified previously, and phaJ4 _Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4_Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4_Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1_Pp was suggested to be different from that of PhaJ1_Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity.     

Antibiofilm activity of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> against cystic fibrosis clinical isolate <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Respiratory tract and device associated infections caused by biofilm forming Pseudomonas aeruginosa play a primary role in the pathogenesis and prognosis of cystic fibrosis (CF) diseases. The biofilm formed by these pathogens attributes to the antibiotic resistance and protection from host immune response. Once established, the pathogens respond poorly to therapeutic agents. Recently medicinal plants are largely explored as potential source of bioactive agents. In this context the present study reports the antibiofilm activity of the folkloric medicinal plant Andrographis paniculata against biofilm forming CF causative Pseudomonas aeruginosa isolated from CF sputum. P. aeruginosa was also assessed for their growth and development of the biofilm, phylogenetic relationship and antibiotic susceptibility. Antibiogram of the strains indicated that they were resistant to more than one antibiotic. Six extracts of A. paniculata showed significant antibiofilm activity. P. aeruginosa strains, KMS P03 and KMS P05, were found to be maximally inhibited by the methanol extract to an extent of 88.6 and 87.5% respectively. This is the first report on antibiofilm activity of A. paniculata extracts, and our results indicate scope for development of complementary medicine for biofilm associated infections.     

Over-expression of <Emphasis Type="Italic">CONSTANS-LIKE 5</Emphasis> can induce flowering in short-day grown <Emphasis Type="Italic">Arabidopsis</Emphasis>

To ensure that the initiation of flowering occurs at the correct time of year, plants need to integrate a diverse range of external and internal signals. In Arabidopsis, the photoperiodic flowering pathway is controlled by a set of regulators that include CONSTANS (CO). In addition, Arabidopsis plants also have a family of genes with homologies to CO known as CO-LIKE (COL) about which relatively little is known. In this paper, we describe the regulation and interactions of a novel member of the family, COL5. The expression of COL5 is under circadian and diurnal regulation, but COL5 itself does not appear to affect circadian rhythms. COL5, like CO, is regulated by GIGANTEA. Furthermore, COL5 is expressed in the vascular tissue. Using COL5 over-expressing lines we show that, under short days, constitutive expression of COL5 affects flowering time and the expression of the floral integrator genes, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO 1. Constitutive expression of COL5 partially suppresses the late flowering phenotype of the co-mutant plants. However, plants with loss of COL5 function do not show altered flowering. Taken together, our results suggest that COL5 has COL activity, but may either not have a role in regulating flowering in wild-type plants or may act redundantly with other flowering regulators. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

Identification and Characterization of <Emphasis Type="Italic">sanH</Emphasis> and <Emphasis Type="Italic">sanI</Emphasis> Involved in the Hydroxylation of Pyridyl Residue During Nikkomycin Biosynthesis in <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis>

Nikkomycins are highly potent inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned from Streptomyces asochromogenes. Two cytochrome P450 monooxygenase genes (sanQ, sanH) and one ferredoxin gene (sanI) were found in the cluster. It was reported that SanQ is involved in the hydroxylation of l-His, a key step in 4-formyl-4-imidazolin-2-one base biosynthesis. Here, we have studied the function of sanH and sanI. Disruption of sanH abolished the production of nikkomycin X and Z, but it accumulated one dominant component nikkomycin Lx, which is the nikkomycin X analog lacking the hydroxy group at the pyridyl residue. The sanI disruption mutant accumulated predominantly nikkomycin Lx in addition to nikkomycin X and Z. The nikkomycin production profile of the sanH and sanI double disruption mutant was the same as that of the sanH disruption mutant. These results confirmed that SanH is essential for the hydroxylation of pyridyl residue in nikkomycin biosynthesis of S. ansochromogenes and first demonstrated that SanI is an effective electron donor for SanH, but not for SanQ in vivo.