Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,protein synthesis,and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of ³⁵S-sulfate and ¹⁴C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.Growth rate constants calculated from increases in cell numbers, protein, and ³⁵S and ¹⁴C in the various fractions indicated nearly balanced growth in A. luteo-violaceus, with constants derived from all biosynthetic parameters agreeing within 5% during the exponential phase. In contrast, protein synthesis and ³⁵S incorporation into residue protein constants were 30% higher than constants derived from cell counts and incorporation of ¹⁴C in P. halodurans. Therefore the cellular protein content P. halodurans varied over a two-fold range, with maximum protein per cell in the late exponential phase. A distinct reduction in the rate constants for total protein and ³⁵S incorporation into residue protein foreshadowed entry into the stationary phase more than one generation before other parameters.Incorporation of ³⁵S-sulfate into residue protein paralleled protein synthesis in both bacteria. The weight percent S in protein agreed well with the composition of an average protein derived from the literature. Sulfur incorporation into protein may be a useful measurement of marine bacterial protein synthesis.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid - CFU colony forming unit     

Biochemical and Functional Characterization of a Eukaryotic-Type Protein Kinase,SpkB, in the Cyanobacterium, <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily (spkA ∼ spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni²⁺ affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg²⁺ and Mn²⁺, but not in Ca²⁺. Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA. Received: 8 June 2002 / Accepted: 5 July 2002     

Interactions between mgr, asp, and polo: asp function modulated by polo and needed to maintain the poles of monopolar and bipolar spindles

We describe genetic interactions between mutations in mgr, asp, and polo, genes required for the correct behaviour of the spindle poles in Drosophila. The phenotype of a polo ¹ mgr double mutant is more similar to mgr than polo ¹ , but the frequency of circular monopolar figures (CMFs) seen with either mutant alone is additive, suggesting that the two gene products are required for independent functions in the formation of bipolar spindles. The asp ^E3 mgr double mutant arrests much earlier in development than either mutant alone, indicative of a strong block to cell proliferation. We discuss whether the lack of microtubular structures in these cells reflects an extended mitotic arrest, or if it is a more direct consequence of the double mutant combination. A polo ¹ asp ^E3 double mutant shows a dramatic synergistic increase in mitotic frequency. The loss of CMFs normally associated with the polo ¹ phenotype suggests that the Asp microtubule-associated protein is required to maintain the structure of spindle poles. We speculate that Asp protein might be a substrate for the serine-threonine protein kinase encoded by polo. Received: 8 August 1998 / Accepted: 13 September 1998     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.     

Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freudii

Summary Chromatographic analyses on a Dowex 50x8 column of tryptic digests of the mutationally altered 50-8 protein component from several erythromycin resistant (ery ^r) mutants of Escherichia coli and Escherichia freundii have been performed. It was found that (1) the difference in the elution profile of the altered components detected with carboxymethyl cellulose column chromatography reflects the difference in their amino acid sequence, (2) the structural change(s) of the 50-8 protein from three E. coli ery ^r mutants examined seems to exist only in the same single peptide fragment and (3) the primary structure of the 50-8(R) protein of E. freundii (ery ^s: wild type) differs from that of E. coli Q13 (ery ^s) and the structural change in 50-8(R) component of E. freundii caused by the ery ^r mutation was found to take place in different peptide fragments from that in which the mutational change of the E. coli 50-8 component occurred.     

Induction of down-regulation of the kinase activities of Mek,p42Erk,p90RSK,and p63SAMK in chicken embryo fibroblast at the late stage of src-induced cellular transformation

Two distinct stages in regulation of protein kinases are detectable upon cellular transformation of CEF induced by pp60^v-src. Upon cellular transformation induced by ts v-src mutants, the kinase activities of Mek and p42^Erk are rapidly induced at the early stage and significantly decreased at the late stage of cellular transformation. In contrast, a novel p63^SAMK is partially activated at the early stage and is fully activated at the late stage of cellular transformation. However, p90^RSK is activated through the entire course of cellular transformation. In this study, I detect a transient down-regulation of p90^RSK activity that is inducible in cultures at the late stage of the src-induced cellular transformation by an increase of extracellular pH value from 7 to 8 and unidentified components in DMEM, but not in cultures which are at the early stage. Concomitant with down-regulation of p90^RSK activity, the kinase activities of Mek, p42^Erk, and p63^SAMK are also down-regulated. Blockage of down-regulation of p90^RSK activity by pretreatment of cells with different phosphatase inhibitors correlates with blockage of the down-regulation of either p42^Erk or p63^SAMK activity. Multiple pathways appear to involve in regulation of p90^RSK activity. The discrepancy in regulation of protein kinase activity between the early and late stages of cellular transformation induced by pp60^src may indicate a change in signaling cascades during the progress of cellular transformation. The induction of the down-regulation event in this study may provide a new approach to investigate the regulation not only of protein kinases but also phosphatases in transformed cells. © 1996 Wiley Liss, Inc.     

Effects of cycloheximide,d-threo-chloramphenicol,erythromycin and actinomycin D on De-novo synthesis of cytoplasmic and mitochondrial proteins in the cotyledons of germinating pea seeds

Summary Inhibitors of, and radioactive substrates for, protein synthesis were introduced into germinating pea (Pisum sativum L.) seeds, and protein synthesis was allowed to proceed in vivo. Subsequent analyses of subcellular fractions showed the following: Cycloheximide strongly inhibited the incorporation of [¹⁴C]leucine into both mitochondrial and cytoplasmic proteins. d-Threo-chloramphenicol and erythromycin did not affect cytoplasmic protein synthesis, but partially inhibited mitochondrial protein synthesis. These results suggest that most of the new mitochondrial proteins were originally synthesized in the cytoplasm. Actinomycin D did not appreciably affect the initial incorporation of [¹⁴C]leucine into either mitochondrial or cytoplasmic proteins, suggesting that information (mRNA) concerning the initially synthesized proteins may be present in the quiescent seeds. The lack of appreciable incorporation of [³H]thymidine into mitochondrial DNA supported our previons report that mitochondria may not be synthesized de novo in pea cotyledons.     

Growth and Protein Content in Colletotrichum circinans, Fusarium solani and Rhizoctonia solani in Liquid Culture

The phytopathogenic fungi Colletotrichum circinans, Fusarium solani, and Rhizoctonia solani were incubated in aerated (0, 0.5, 1 dm³ min^–1) potato dextrose broth (PDB) or Czapek-Dox broth (CDB), under 0-, 12- or 24-h photoperiods. Greater dry mass was produced in PDB. Higher air flows improved dry mass of F. solani and R. solani. The 24-h photoperiod improved F. solani dry mass. Except for F. solani, which was not affected, incubation in PDB increased protein content. The no air treatment increased protein content in F. solani, 0.5 dm³ min^–1 produced the highest protein content in R. solani, but air flow-rate did not affect C. circinans. Incubation in the dark produced the lowest protein content in C. circinans, the highest under the 24-h photoperiod for R. solani, and photoperiod did not affect protein content in F. solani. Protein content in R. solani, incubated in CDB, was lowest at the 0 dm³ min^–1 air flow at all photoperiods. Molecular masses of most proteins were under 30 kDa, and numbers of bands in SDS-PAGE gels varied with air flow. In CDB, especially under 12- or 24-h photoperiods, proteins in F. solani were between 1.6 and 310 kDa. For R. solani in PDB, at 0.5 dm³ min^–1 air flow and 12-h light, proteins were between 6 and 81 kDa.     

Host derived inflammatory phospholipids regulate rahU (PA0122) gene, protein, and biofilm formation in Pseudomonas aeruginosa

This study describes the role of “inflammatory” oxidized (Ox) phospholipids in regulation of rahU (PA0122) expression and biofilm formation in Pseudomonas aeruginosa (383) wild type (rahU⁺) and rahU mutant (rahU⁻) strains. Functional analysis of RahU protein from P. aeruginosa in presence of Ox-phospholipids show: (a) LysoPC modulates RahU gene/and protein expression in rahU⁺ cells; (b) rahU promoter activity is increased by lysoPC and inhibited by PAPC, Ox-PAPC and arachidonic acid; the latter inhibitory effect can be reversed by lysoPC, which was enzymatically derived from PAPC; (c) biofilm formation increased in rahU⁻ cells as compared to rahU⁺; and (d) inhibition of rahU promoter activity by PAPC and AA (but not lysoPC) showed significantly augmented biofilm formation in rahU⁺ but not in rahU⁻ cells. This study shows that host derived Ox-phospholipids affect P. aeruginosa-rahU gene and protein expression, which in turn modulates biofilm formation. The accompanying paper describes the role of RahU protein in eukaryotic-host cells.     

Taste,movement, and death: Varying effects of new prospero mutants during Drosophila development

The PGal4 transposon inserted upstream of the pan‐neural gene prospero (pros) causes several neural and behavioral defects in the Voila¹ strain. The precise excision of the transposon simultaneously rescued all these defects whereas its unprecise excision created new pros^V alleles, including the null allele pros^V17. Here, we describe the relationship between the genetic structure of pros locus, larval locomotion, and larval gustatory response. These two behaviors showed varying degrees of variation depending upon the pros allele. We also found a good relation between behavioral alteration, the level of Pros protein in the embryo, and the degree of disorganization in the larval neuromuscular junction. These data suggest that the complete development of the nervous system requires a full complement of Pros, and that a gradual decrease in the levels of this protein can proportionally alter the development and the function of the nervous system. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 1–13, 2003     

A kinetic analysis of cell division, and induction and stability of recA protein in U.V. Irradiated ion+ and ion-strains of Escherichia coli K12.

Summary Kinetic analysis of induction of recA protein synthesis after U.V. irradiation does not show correspondence with the kinetics of division inhibition in lon ⁺ and lon ^- strains, but there is correlation between induction and DNA repair activity. Protein X is stable and identical in both lon ⁺ and lon ^- strains. When the induction of recA protein after U.V. is drastically reduced by rifampicin treatment, no effect on the kinetics of division inhibition is observed.     

Cloning, Characterization, and Functional Expression in Escherichia coli of argH Encoding Argininosuccinate Lyase in the Cyanobacterium Nostoc sp. Strain PCC 73102

A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp. strain PCC 73102. The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da. Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms. In an Escherichia coliΔargH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine. Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea · min⁻¹· (mg protein)⁻¹. Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in native-PAGE, suggesting that the native protein is a tetramer. Received: 6 December 2000 / Accepted: 9 February 2001     

1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum

We report the ¹H, ¹³C, and ¹⁵N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, isolated from the acidophilic gram-negative facultatively anaerobic metal-reducing alphaproteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c ₂ and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on ¹³C- and ¹⁵N- labeled substrates.     

Overexpression, on-column refolding and isotopic labeling of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily

A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of βγ-crystallin superfamily. This hypothetical protein contains a putative βγ-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (¹⁵N/¹³C) labeled protein with high yield for NMR studies is reported. The uniformly ¹⁵N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [¹⁵N]–[¹H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.     

Sequence-specific 1H, 13C, and 15N backbone assignment of Psb27 from Synechocystis PCC 6803

Photosystem II (PSII) is a large membrane protein complex that uses light to split water into molecular oxygen, protons, and electrons. Here we report the ¹H, ¹⁵N and ¹³C backbone chemical shift assignments for the Psb27 protein of Photosystem II from Synechocystis PCC 6803. These assignments will now provide the basis for the structural analysis of the Psb27 protein.     

Processing, Disulfide Pattern, and Biological Activity of a Sugar Beet Defensin, AX2, Expressed in Pichia pastoris

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca²⁺ than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.     

1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I

Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled ¹³C/¹⁵N recombinant protein was produced in E. coli. Here we report the complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of Stn I at pH 4.0 and 25°C (BMRB No. 15927).