Wide variation in the cyanobacterial complement of presumptive penicillin-binding proteins

A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase–transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A high-molecular-weight PBPs that are required for the functional differentiation of aerobically diazotrophic heterocysts, indicating that some members of this class of PBPs are required specifically for cellular developmental processes.     

Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels.     

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more ¹³NO₃⁻ into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.     

in-silico analysis suggests alterations in the function of XisA protein as a possible mechanism of butachlor toxicity in the nitrogen fixing cyanobacterium Anabaena sp. PCC 7120

Butachlor, a commonly used herbicide adversely affects the nitrogen fixing capability of Anabaena, an acclaimed nitrogen fixer in the Indian paddy fields. The nitrogen fixation in Anabaena is triggered by the excision of nifD element by xisA gene leading to rearrangement of nifD forming nifHDK operon in the heterocyst of Anabaena sp. PCC7120. Functional elucidation adjudged through in-silico analysis revealed that xisA belongs to integrase family of tyrosine recombinase. The predicted functional partners with XisA protein that have shown cooccurence with this protein in a network are mainly hypothetical proteins with unknown functions except psaK1 whose exact function in photosystem I is not yet known. The focus of this study was to find out the relation between XisA and butachlor using in-silico approaches. The XisA protein was modeled and its active sites were identified. Docking studies revealed that butachlor binds at the active site of XisA protein hampering its excision ability vis-à-vis nif genes in Anabaena sp. PCC7120. This study reveals that butachlor is not directly involved in hampering the nitrogen fixing ability of Anabaena sp. PCC7120 but by arresting the excision ability of XisA protein necessary for the functioning of nif gene and nitrogen fixation.     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

HcwA, an autolysin, is required for heterocyst maturation in Anabaena sp. strain PCC 7120.

In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N(2). Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA in Anabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomal hepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resembles N-acetylmuramoyl-L-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of hepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.     

Role of the all1549 (ana-rsh) gene, a relA/spoT homolog, of the Cyanobacterium Anabaena sp. PCC7120

The role of a single relA/spoT homolog all1549 (designated hereafter as ana-rsh) of the cyanobacterium Anabaena sp. PCC7120 was investigated. The complementation test in Escherichia coli showed that the protein encoded by ana-rsh possesses guanosine tetraphosphate (p)ppGpp-synthase/hydrolase activity. Under laboratory growth conditions, a low level of ppGpp was detected in Anabaena sp. PCC7120 and the loss of ana-rsh was lethal. Amino acid starvation induced ppGpp accumulation to an appropriate level, and nitrogen deficiency did not alter the ppGpp concentration in Anabaena cells. These data suggest that ana-rsh is required for cell viability under normal growth conditions and involved in the (p)ppGpp-related stringent response to amino acid deprivation, but not related to heterocyst formation and nitrogen fixation of Anabaena sp. PCC7120.     

Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H₂ producers.     

Characterization of a DUF820 family protein Alr3200 of the cyanobacterium <Emphasis Type="BoldItalic">Anabaena</Emphasis> sp. strain PCC7120

The hypothetical protein ‘Alr3200’ of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterial species. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have a DNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a specific activity of 8.62×10⁴ Kunitz Units (KU) mg^?1 protein. Circular dichroism analysis predicted Alr3200 to have ~40% β-strands and ~9% α-helical structures. Anabaena PCC7120 inherently expressed Alr3200 at very low levels, and its overexpression had no significant effect on growth of Anabaena under control conditions. However, Analr3200 ⁺, the recombinant Anabaena strain overexpressing Alr3200, exhibited zero survival upon exposure to 6 kGy of γ-radiation, which is the LD₅₀ for wild type Anabaena PCC7120 as well as the vector control recombinant strain, AnpAM. Comparative analysis of the two recombinant Anabaena strains suggested that it is not the accumulated Alr3200 per se, but its possible interactions with the radiation-induced unidentified DNA repair proteins of Anabaena, which hampers DNA repair resulting in radiosensitivity.     

The Hypothetical Protein ‘All4779’, and Not the Annotated ‘Alr0088’ and ‘Alr7579’ Proteins,Is the Major Typical Single-Stranded DNA Binding Protein of the Cyanobacterium,Anabaena sp. PCC7120

Single-stranded DNA binding (SSB) proteins are essential for all DNA-dependent cellular processes. Typical SSB proteins have an N-terminal Oligonucleotide-Binding (OB) fold, a Proline/Glycine rich region, followed by a C-terminal acidic tail. In the genome of the heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120, alr0088 and alr7579 are annotated as coding for SSB, but are truncated and have only the OB-fold. In silico analysis of whole genome of Anabaena sp. strain PCC7120 revealed the presence of another ORF ‘all4779’, annotated as a hypothetical protein, but having an N-terminal OB-fold, a P/G-rich region and a C-terminal acidic tail. Biochemical characterisation of all three purified recombinant proteins revealed that they exist either as monomer or dimer and bind ssDNA, but differently. The All4779 bound ssDNA in two binding modes i.e. (All4779)₃₅ and (All4779)₆₆ depending on salt concentration and with a binding affinity similar to that of Escherichia coli SSB. On the other hand, Alr0088 bound in a single binding mode of 50-mer and Alr7579 only to large stretches of ssDNA, suggesting that All4779, in all likelihood, is the major typical bacterial SSB in Anabaena. Overexpression of All4779 in Anabaena sp. strain PCC7120 led to enhancement of tolerance to DNA-damaging stresses, such as γ-rays, UV-irradiation, desiccation and mitomycinC exposure. The tolerance appears to be a consequence of reduced DNA damage or efficient DNA repair due to increased availability of All4779. The ORF all4779 is proposed to be re-annotated as Anabaena ssb gene.     

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N₂‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.     

Enhanced Resistance to UV-B Radiation in Anabaena sp. PCC 7120 (Cyanophyceae) by Repeated Exposure

In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N₂-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats.     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

Heterocyst patterns without patterning proteins in cyanobacterial filaments

We have quantitatively modeled heterocyst differentiation after fixed nitrogen step-down in the filamentous cyanobacterium Anabaena sp. PCC 7120 without lateral inhibition due to the patterning proteins PatS or HetN. We use cell growth and division together with fixed-nitrogen dynamics and allow heterocysts to differentiate upon the local exhaustion of available fixed nitrogen. Slow transport of fixed nitrogen along a shared periplasmic space allows for fast growing cells to differentiate ahead of their neighbors. Cell-to-cell variability in growth rate determines the initial heterocyst pattern. Early release of fixed nitrogen from committed heterocysts allows a significant fraction of vegetative cells to be retained at later times. We recover the experimental heterocyst spacing distributions and cluster size distributions of Khudyakov and Golden [Khudyakov, I.Y., Golden, J.W., 2004. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16040-16045].     

The structural nif genes of four symbiotic Anabaena azollae show a highly conserved physical arrangement

Cloned DNA from Anabaena sp. PCC 7120 was used to determine the distribution of restriction sites around NifHDK genes of endosymbionts extracted from Azolla species. Although many of the restriction sites of the symbiotic Anabaena nifHDK genes differed from those of the free-living Anabaeba sp. PCC 7120, three apparently identical restriction sites were found in and around the nifD region. The arrangement of A. azollae and Anabaena sp. PCC 7120 nif H,D,K genes appears similar, with nifH and nifD linked and nifK some distance away from nifD. The A. azollae nifHDK genes appear strongly conserved among the Azolla species examined, regardless of the geographical origin of the ferns.