Sll1252 Coordinates Electron Transport between Plastoquinone and Cytochrome b6/f Complex in Synechocystis PCC 6803.

A mutant, Δsll1252ins, was generated to functionally characterize Sll1252. Δsll1252ins exhibited a slow-growth phenotype at 70 µmol photons m-2 s-1 and glucose sensitivity. In Δsll1252ins, the rate of PSII activity was not affected, whereas the whole chain electron transport activity was reduced by 45%. The inactivation of sll1252 led to the upregulation of genes, which were earlier reported to be induced in DBMIB-treated wild-type, suggesting that Sll1252 may be involved in electron transfer from the reduced-PQ pool to Cyt b6/f. The inhibitory effect of DCMU on PSII activity was similar in both wild-type and Δsll1252ins. However, the concentration of DBMIB for 50% inhibition of whole chain electron transport activity was 140 nM for Δsll1252ins and 300 nM for wild-type, confirming the site of action of Sll1252. Moreover, the elevated level of the reduced-PQ pool in Δsll1252ins supports that Sll1252 functions between the PQ pool and Cyt b6/f. Interestingly, we noticed that Δsll1252ins reverted to wild-type phenotype by insertion of natural transposon, ISY523, at the disruption site. Δsll1252-Ntrn, expressing only the C-terminal region of Sll1252, exhibited a slow-growth phenotype and disorganized thylakoid structure compared to wild-type and Δsll1252-Ctrn (expressing only the N-terminal region). Collectively, our data suggest that Sll1252 regulates electron transfer between the PQ pool and the Cyt b6/f complex in the linear photosynthetic electron transport chain via coordinated function of both the N- and C-terminal regions of Sll1252.

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides.

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance, and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival, and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT-PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature, and surface association strongly affect the expression of different polysaccharides under the control of the signalling protein genes ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides play a role in mediating leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection.

Comparative genome analysis of Alkalihalobacillus okhensis Kh10-101 T reveals insights into adaptive mechanisms for halo-alkali tolerance.

Alkalihalobacillus okhensis is a halo-alkaliphile with optimal growth at pH 10 and 5% NaCl. Phylogenetic analysis revealed habitat-dependent segregation of Bacilli, with all the alkalihalophiles forming a separate clade. It uses acidification of the external medium and pH-dependent cell wall reinforcement to survive sodic environments. Interestingly, comparative genome analysis revealed the genome encodes surface proteins with a high proportion of acidic amino acids compared to their orthologs of B. subtilis, a piece of direct evidence for adaptive evolution. It has a relatively higher number of genes involved in the metabolism of osmolytes and sodium-dependent transporters when compared to B. subtilis. Growth of Alkalihalobacillus okhensis strain Kh10-101 T (hereafter A. okhensis) is Na+ dependent, with a minimum of 4% NaCl at neutral pH, but 0.5% NaCl is enough at pH 10. It tolerated a sudden increase in salt concentration and exhibited an elongated phenotype but could not tolerate a sudden pH shift from 7 to 11. The cell envelope got damaged, confirming that the pH regulation through cell wall reinforcement is key to survival at a high-pH condition. We report for the first time a comprehensive genome analysis of Bacilli to delineate the mechanisms evolved for adaptation to halo-alkaline conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02938-x.

Characterization of the transient fluorescence wave phenomenon that occurs during H2 production in Chlamydomonas reinhardtii.

The redox state of the plastoquinone (PQ) pool in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cells was studied using single flash-induced variable fluorescence decay kinetics. During H2 production, the fluorescence decay kinetics exhibited an unusual post-illumination rise of variable fluorescence, giving a wave-like appearance. The wave showed the transient fluorescence minimum at ~60 ms after the flash, followed by a rise, reaching the transient fluorescence maximum at ~1 s after the flash, before decaying back to the initial fluorescence level. Similar wave-like fluorescence decay kinetics have been reported previously in anaerobically incubated cyanobacteria but not in green algae. From several different electron and proton transfer inhibitors used, polymyxin B, an inhibitor of type II NAD(P)H dehydrogenase (NDA2), had the effect of eliminating the fluorescence wave feature, indicating involvement of NDA2 in this phenomenon. This was further confirmed by the absence of the fluorescence wave in the Δnda2 mutant lacking NDA2. Additionally, Δnda2 mutants have also shown delayed and diminished H2 production (only 23% if compared with the wild type). Our results show that the fluorescence wave phenomenon in C. reinhardtii is observed under highly reducing conditions and is induced by the NDA2-mediated electron flow from the reduced stromal components to the PQ pool. Therefore, the fluorescence wave phenomenon is a sensitive probe for the complex network of redox reactions at the PQ pool level in the thylakoid membrane. It could be used in further characterization and improvement of the electron transfer pathways leading to H2 production in C. reinhardtii.

The Ssl2245-Sll1130 Toxin-Antitoxin System Mediates Heat-induced Programmed Cell Death in Synechocystis sp. PCC6803.

Two putative heat-responsive genes, ssl2245 and sll1130, constitute an operon that also has characteristics of a toxin-antitoxin system, thus joining several enigmatic features. Closely related orthologs of Ssl2245 and Sll1130 exist in widely different bacteria, which thrive under environments with large fluctuations in temperature and salinity, among which some are thermo-epilithic biofilm-forming cyanobacteria. Transcriptome analyses revealed that the clustered regularly interspaced short palindromic repeats (CRISPR) genes as well as several hypothetical genes were commonly up-regulated in Δssl2245 and Δsll1130 mutants. Genes coding for heat shock proteins and pilins were also induced in Δsll1130 We observed that the majority of cells in a Δsll1130 mutant strain remained unicellular and viable after prolonged incubation at high temperature (50 °C). In contrast, the wild type formed large cell clumps of dead and live cells, indicating the attempt to form biofilms under harsh conditions. Furthermore, we observed that Sll1130 is a heat-stable ribonuclease whose activity was inhibited by Ssl2245 at optimal temperatures but not at high temperatures. In addition, we demonstrated that Ssl2245 is physically associated with Sll1130 by electrostatic interactions, thereby inhibiting its activity at optimal growth temperature. This association is lost upon exposure to heat, leaving Sll1130 to exhibit its ribonuclease activity. Thus, the activation of Sll1130 leads to the degradation of cellular RNA and thereby heat-induced programmed cell death that in turn supports the formation of a more resistant biofilm for the surviving cells. We suggest to designate Ssl2245 and Sll1130 as MazE and MazF, respectively.

Draft genome sequence of Bacillus okhensis Kh10-101T, a halo-alkali tolerant bacterium from Indian saltpan.

We report the 4.86-Mb draft genome sequence of Bacillus okhensis strain Kh10-101T, a halo-alkali tolerant rod shaped bacterium isolated from a salt pan near port of Okha, India. This bacterium is a potential model to study the molecular response of bacteria to salt as well as alkaline stress, as it thrives under both high salt and high pH conditions. The draft genome consist of 4,865,284 bp with 38.2% G + C, 4952 predicted CDS, 157 tRNAs and 8 rRNAs. Sequence was deposited at DDBJ/EMBL/GenBank under the project accession JRJU00000000.

A novel transcriptional regulator, Sll1130, negatively regulates heat-responsive genes in Synechocystis sp. PCC6803.

A conserved hypothetical protein, Sll1130, is a novel transcription factor that regulates the expression of major heat-responsive genes in Synechocystis sp. PCC6803. Synechocystis exhibited an increased thermotolerance due to disruption of sll1130. Δsll1130 cells recovered much faster than wild-type cells after they were subjected to heat shock (50°C) for 30 min followed by recovery at 34°C for 48 h. In Δsll1130 cultures, 70% of the cells were viable compared with the wild-type culture in which only 30% of the cells were viable. DNA microarray analysis revealed that in Δsll1130, expression of the heat-responsive genes such as htpG, hspA, isiA, isiB and several hypothetical genes were up-regulated. Sll1130 binds to a conserved inverted-repeat (GGCGATCGCC) located in the upstream region of the above genes. In addition, both the transcript and protein levels of sll1130 were immediately down-regulated upon shift of wild-type cells from 34 to 42°C. Collectively the results of the present study suggest that Sll1130 is a heat-responsive transcriptional regulator that represses the expression of certain heat-inducible genes at optimum growth temperatures. Upon heat shock, a quick drop in the Sll1130 levels leads to de-repression of the heat-shock genes and subsequent thermal acclimation. On the basis of the findings of the present study, we present a model which describes the heat-shock response involving Sll1130.

RNA helicase, CrhR is indispensable for the energy redistribution and the regulation of photosystem stoichiometry at low temperature in Synechocystis sp. PCC6803.

We investigated the role of a cold-inducible and redox-regulated RNA helicase, CrhR, in the energy redistribution and adjustment of stoichiometry between photosystem I (PSI) and photosystem II (PSII), at low temperature in Synechocystis sp. PCC 6803. The results suggest that during low temperature incubation, i.e., when cells are shifted from 34°C to 24°C, wild type cells exhibited light-induced state transitions, whereas the mutant deficient in CrhR failed to perform the same. At low temperature, wild type cells maintained the plastoquinone (PQ) pool in the reduced state due to enhanced respiratory electron flow to the PQ pool, whereas in ∆crhR mutant cells the PQ pool was in the oxidized state. Wild type cells were in state 2 and ∆crhR cells were locked in state 1 at low temperature. In both wild type and ∆crhR cells, a fraction of PSI trimers were changed to PSI monomers. However, in ∆crhR cells, the PSI trimer content was significantly decreased. Expression of photosystem I genes, especially the psaA and psaB, was strongly down-regulated due to oxidation of downstream components of PQ in ∆crhR cells at low temperature. We demonstrated that changes in the low temperature-induced energy redistribution and regulation of photosystem stoichiometry are acclimatization responses exerted by Synechocystis cells, essentially regulated by the RNA helicase, CrhR, at low temperature.

An RNA helicase, CrhR, regulates the low-temperature-inducible expression of heat-shock genes groES, groEL1 and groEL2 in Synechocystis sp. PCC 6803.

The crhR gene for RNA helicase, CrhR, was one of the most highly induced genes when the cyanobacterium Synechocystis sp. PCC 6803 was exposed to a downward shift in ambient temperature. Although CrhR may be involved in the acclimatization of cyanobacterial cells to low-temperature environments, its functional role during the acclimatization is not known. In the present study, we mutated the crhR gene by replacement with a spectinomycin-resistance gene cassette. The resultant DeltacrhR mutant exhibited a phenotype of slow growth at low temperatures. DNA microarray analysis of the genome-wide expression of genes, and Northern and Western blotting analyses indicated that mutation of the crhR gene repressed the low-temperature-inducible expression of heat-shock genes groEL1 and groEL2, at the transcript and protein levels. The kinetics of the groESL co-transcript and the groEL2 transcript after addition of rifampicin suggested that CrhR stabilized these transcripts at an early phase, namely 5-60 min, during acclimatization to low temperatures, and enhanced the transcription of these genes at a later time, namely 3-5 h. Our results suggest that CrhR regulates the low-temperature-inducible expression of these heat-shock proteins, which, in turn, may be essential for acclimatization of Synechocystis cells to low temperatures.