Identification and characterization of the serine/threonine protein kinases in Bifidobacterium

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.     

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.     

Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803

The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA⁺, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.     

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca²⁺ ions. The inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca²⁺-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent STPK reduced the Ca²⁺-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.     

PknE, a serine/threonine protein kinase from Mycobacterium tuberculosis has a role in adaptive responses

Serine/threonine protein kinases (STPK) play a major role in the physiology and pathogenesis of Mycobacterium tuberculosis. Here, we have examined the role of pknE, a STPK in the adaptive responses of M. tuberculosis using a deletion mutant ΔpknE. The survival of ΔpknE was assessed in the presence of stress (pH, surfactant and cell wall–damaging agents) and anti-tuberculosis drugs. ΔpknE had a defective growth in pH 7.0 and lysozyme (a cell wall–damaging agent) with better survival in pH 5.5, SDS and kanamycin (a second-line anti-tuberculosis drug). Furthermore, ΔpknE was reduced in cell size during growth in liquid media and exhibited hypervirulence in a guinea pig model of infection. In conclusion, our data suggest that pknE plays a role in adaptive response of M. tuberculosis regulating cellular integrity and survival.     

Identification and Characterization of the ictA/ndhL Gene Product Essential to Inorganic Carbon Transport of Synechocystis PCC6803

The ictA gene, renamed ndhL in this paper, essential to inorganic carbon transport of Synechocystis PCC6803, was expressed in Eschericia coli as a fusion protein with glutathione S-transferase. An antibody was raised against this fusion protein. Western analysis of the thylakoid membrane of wild-type (WT) Synechocystis revealed that a protein with an apparent molecular mass of 6.7 kilodaltons cross-reacted with this antibody. No immunoreactive protein was present in the thylakoid membranes of the Synechocystis mutants, RKb and M9, which have defects in the ictA/ndhL gene, or in the cytoplasmic membranes of the WT and mutant cells. Thus, the protein reacted with the antibody is the ictA gene product (IctA) and is localized in the thylakoid membrane of WT cells. IctA was absent in the thylakoid membranes of the M55 mutant, in which the ndhB gene is inactivated, and was poorly immunostained in the membranes of the mutants (M-ndhC and M-ndhK) constructed by inactivating the ndhC and ndhK genes of WT Synechocystis, respectively. The carbon dioxide uptake activity was nearly zero in M-ndhK and was about 40% of the activity of WT cells in M-ndhC. The RKb, M-ndhC, and M-ndhK mutants were unable to grow or grew very slowly under photoheterotrophic conditions. These results indicated that NADH dehydrogenase is essential to inorganic carbon transport and photoheterotrophic growth of Synechocystis and that IctA is one of the subunits of this enzyme.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System

RNA is an essential component for the enzymic conversion of glutamate to δ-aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA^Glu(UUC), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA^Glu(UUC) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA^Glu(UUC) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [³H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA^Glu(UUC) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA^Glu(UUC) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA^Glu(UUC) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [³H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.     

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P_trc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P_A1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.     

Using photosystem I as a reporter protein for C analysis in a coculture containing cyanobacterium and a heterotrophic bacterium

¹³C metabolism analysis of a microbial community is often hindered by the time-consuming and complicated separation procedure for a single species. However, a “reporter protein,” produced uniquely by one cell type, retains ¹³C fingerprint information in microbial consortia. This study describes the use of photosystem I (PSI), a multi-subunit protein complex universally found in oxygenic phototrophs, as a reliable reporter protein to probe microalgal metabolism (i.e., cyanobacterium Synechocystis sp. PCC 6803) in a mixed culture with heterotrophic bacteria (i.e., Escherichia coli). We demonstrate that efficient purification of PSI and subsequent ¹³C-based amino acid analyses may decipher photomixotrophic metabolism of Synechocystis 6803 in the coculture. This study also indicates that a supplement of NaHCO₃ at high concentration could significantly improve the robustness of cyanobacterial growth against bacterial contamination.     

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negative-stain transmission electron microscopy, a ∼30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the Δsll1951 strain. Instead, the Δsll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the Δsll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the Δsll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the Δsll1951 strain was similar to that of the wild-type strain, the viability of the Δsll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.     

Induction of programmed lysis in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> culture by the inhibitors of eukaryotic type serine/threonine protein kinases

Programmed death (PD) of the mycelium of Streptomyces lividans, namely, its delayed lysis in response to treatment with indolylmaleimide derivatives, which inhibit actinobacterial serine/threonine protein kinases (STPK), is described. Delayed lysis of mycelial cell was accompanied by DNA damage similar to PD in differentiating S. lividans mycelium. Two-dimensional electrophoresis and mass spectrometry were used to identify proteins up-regulated by a PD-inducing STPK inhibitor. Most of these proteins are known to be implicated in responses to various stress stimuli. Thus, our model of delayed cell lysis of actinobacteria upon STPK inhibition may serve for unveiling the molecular mechanisms of bacterial PD and for antimicrobial drug design.     

Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis

Many Gram-positive bacteria coordinate cellular processes by signaling through Ser/Thr protein kinases (STPKs), but the architecture of these phosphosignaling cascades is unknown. To investigate the network structure of a prokaryotic STPK system, we comprehensively explored the pattern of signal transduction in the Mycobacterium tuberculosis Ser/Thr kinome. Autophosphorylation is the dominant mode of STPK activation, but the 11 M. tuberculosis STPKs also show a specific pattern of efficient cross-phosphorylation in vitro. The biochemical specificity intrinsic to each kinase domain was used to map the provisional signaling network, revealing a three-layer architecture that includes master regulators, signal transducers, and terminal substrates. Fluorescence microscopy revealed that the STPKs are specifically localized in the cell. Master STPKs are concentrated at the same subcellular sites as their substrates, providing additional support for the biochemically defined network. Together, these studies imply a branched functional architecture of the M. tuberculosis Ser/Thr kinome that could enable horizontal signal spreading. This systems-level approach provides a biochemical and spatial framework for understanding Ser/Thr phospho-signaling in M. tuberculosis, which differs fundamentally from previously defined linear histidine kinase cascades.     

Eukaryotic protein kinases in cyanobacteria

This review presents the data on the role of eukaryotic-like serine/threonine protein kinases in the members of various groups of cyanobacteria. Information is provided for the two most studied model species (Anabaena and Synechocystis), differing in their morphology and ecophysiological features, and covers the entire period of study of this group of enzymes in cyanobacteria.     

A role for mrgA, a DPS family protein, in the internal transport of Fe in the cyanobacterium Synechocystis sp. PCC6803

The mrgA protein of the cyanobacterium Synechocystis sp. PCC6803 is a member of the DPS Fe storage protein family. The physiological role of this protein was studied using a disruption mutant in the mrgA gene (slr1894) and by measuring intracellular Fe quotas, 77K chlorophyll fluorescence and growth rates. It was found that the deletion of the mrgA gene did not impair the Fe storage capacity, as the intracellular Fe quotas of the ΔmrgA cells were comparable to those of the wild type. Furthermore, the cellular response to decreasing external Fe concentrations, as detected by the emergence of the CP43′ 77K fluorescence band, was similar in wild type and mutant cultures. On the other hand, a considerable slow down in the growth rate of ΔmrgA cultures was observed upon transfer from Fe replete to Fe depleted medium, indicating impeded utilization of the plentiful intracellular Fe. Based on these results, we suggest that mrgA plays an important role in the transport of intracellular Fe from storage (within bacterioferritins) to biosynthesis of metal cofactors throughout the cell's growth.     

Proximity Labeling Facilitates Defining the Proteome Neighborhood of Photosystem II Oxygen Evolution Complex in a Model Cyanobacterium

Ascorbate peroxidase (APEX)-based proximity labeling coupled with mass spectrometry has a great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO. In total, 38 integral membrane proteins (IMPs) and 93 luminal proteins were identified as proximal to the OEC. A significant portion of these proteins are involved in PS II assembly, maturation, and repair, while the majority of the rest were not previously implicated with PS II. The IMPs include subunits of PS II and cytochrome b₆/f, but not of photosystem I (except for PsaL) and ATP synthases, suggesting that the latter two complexes are spatially separated from the OEC with a distance longer than the APEX2 labeling radius. Besides, the topologies of six IMPs were successfully predicted because their lumen-facing regions exclusively contain potential APEX2 labeling sites. The luminal proteins include 66 proteins with a predicted signal peptide and 57 proteins localized also in periplasm, providing important targets to study the regulation and selectivity of protein translocation. Together, we not only developed a robust workflow for the application of APEX2-based proximity labeling in Synechocystis and showcased the feasibility to define the neighborhood proteome of an important protein complex with a short radius but also discovered a set of the proteins that potentially interact with and regulate PS II structure and function.     

Global Landscape of Native Protein Complexes in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803(hereafter: Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Although known as the first fully sequenced phototrophic organism, Synechocystis still has almost half of its proteome without functional annotations. In this study, by using co-fractionation coupled with liquid chromatographytandem mass spectrometry(LC-MS/MS), we define 291 multi-protein complexes, encompassing24,092 protein±protein interactions(PPIs...     

The Cyanobacterium Synechocystis sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N⁶-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C⁵-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5′-CGATCG-3′, corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation SynMI (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.