Comparative analysis reveals distinctive genomic features of Taiwan hot-spring cyanobacterium Thermosynechococcus sp. TA-1.

Thermosynechococcus is a genus of thermophilic unicellular cyanobacteria that dominates microbial mats in Asian non-acidic hot springs. These cyanobacteria are the major primary producers in their ecological niches and are promising sources of thermostable enzymes for biotechnology applications. To improve our understanding of these organisms, we conducted whole-genome sequencing of a novel strain for comparative analysis with other representatives in the same genus. This newly characterized strain, Thermosynechococcus sp. TA-1, was isolated from the Taian hot springs in Taiwan. Analyses based on average nucleotide identity (ANI) and genome-scale phylogeny suggested that TA-1 and another Taiwanese strain CL-1 belong to a novel species-level taxon. Two metagenome-assembled genomes (MAGs) originated from India represent the sister group, and Thermosynechococcus elongatus PKUAC-SCTE542 from China is the next closest lineage. All cultivated strains and MAGs from Japan form a separate monophyletic clade and could be classified into two species-level taxa. Intriguingly, although TA-1 and CL-1 share 97.0% ANI, the genome alignment identified at least 16 synteny breakpoints that are mostly associated with transposase genes, which illustrates the dynamic nature of their chromosomal evolution. Gene content comparisons identified multiple features distinct at species- or strain-level among these Thermosynechococcus representatives. Examples include genes involved in bicarbonate transportation, nitric oxide protection, urea utilization, kanamycin resistance, restriction-modification system, and chemotaxis. Moreover, we observed the insertion of type II inteins in multiple genes of the two Taiwanese strains and inferred putative horizontal transfer of an asparagine synthase gene (asnB) associated with exopolysaccharides gene cluster. Taken together, while previous work suggested that strains in this genus share a highly conserved genomic core and no clear genetic differentiation could be linked to environmental factors, we found that the overall pattern of gene content divergence is largely congruent with core genome phylogeny. However, it is difficult to distinguish between the roles of phylogenetic relatedness and geographic proximity in shaping the genetic differentiation. In conclusion, knowledge of the genomic differentiation among these strains provides valuable resources for future functional characterization.

A statistical approach to identify prevalent virulence factors responsible for post-weaning diarrhoeic piglets.

A statistical approach was carried out to identify the prevalent virulence factors responsible for post-weaning diarrhoea (PWD). Healthy piglets' faecal samples and diarrhoeic piglets' rectal swab specimens were secured. Twenty-six (26) and 100 independent enterotoxigenic Escherichia coli (ETEC) strains were subsequently isolated. These strains were assessed utilising polymerase chain reaction to identify the encoding genes of six virulence factors: heat-labile enterotoxin (LT; encoded by eltAB), heat-stable enterotoxin A (STa; encoded by estA), heat-stable enterotoxin B (STb; encoded by estB), enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1; encoded by astA), Shiga toxin 2e (Stx2e; encoded by stx2e), and F18 fimbriae (encoded by fedA). The LT and ST secretions were investigated using enzyme-linked immunosorbent assays. From direct observation, no stx2e was evident in the 126 strains. Among the 26 strains retrieved from the healthy piglets, none harboured fedA or secreted LT; 23% (6/26) secreted ST, and 50% (13/26) carried astA. A statistical regression was applied on the 100 E. coli strains retrieved from the diarrhoeic piglets, where fedA was set as the dependent variable and the enterotoxin secretions were set as the independent variables. The results exhibit that the LT secretion was the only significant factor (P < 0.000 1) correlated to fedA in the diarrhoeic piglets; thus, it is concluded that the prevalent virulence factors for PWD were the ECET strain with F18 fimbriae adhesion and LT secretion, but not astA or stx2e.

Comparative Genomic Analysis of a Novel Strain of Taiwan Hot-Spring Cyanobacterium Thermosynechococcus sp. CL-1.

Thermosynechococcus is a genus of thermophilic unicellular cyanobacteria that are dominant in microbial mats at about 50-65°C in alkaline hot springs of eastern Asia. We used PacBio SMRT Sequencing to sequence the complete genome of a novel strain of thermophilic cyanobacterium, Thermosynechococcus sp. CL-1, isolated from the Chin-Lun hot spring (pH 9.3, 62°C) in Taiwan. Genome-scale phylogenetic analysis and average nucleotide identity (ANI) results suggested that CL-1 is a new species in the genus Thermosynechococcus. Comparative genome analysis revealed divergent genome structures of Thermosynechococcus strains. In addition, the distinct genetic differences between CL-1 and the other Thermosynechococcus strains are related to photosynthesis, transporters, signal transduction, the chaperone/usher system, nitric oxide protection, antibiotic resistance, prokaryotic immunity systems, and other physiological processes. This study suggests that Thermosynechococcus strains have actively acquired many putative horizontally transferred genes from other bacteria that enabled them to adapt to different ecological niches and stressful conditions in hot springs.

Calcium-induced proline accumulation contributes to amelioration of NaCl injury and expression of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

The calcium-mediated proline accumulation is a critical response under NaCl stress and the function of the induced proline as a glutamine synthetase (GS) protectant in greater duckweed was investigated. The plants were treated with solutions containing 100mM NaCl, 200 mM NaCl, 200 mM NaCl plus 10mM CaCl2, or 10mM CaCl2 alone for 4 days. At the end of the experiment, the fronds of inoculum treated with 200 mM NaCl showed the chlorotic effect, higher glutamate dehydrogenase (NADH-GDH) activity and lower GS activity. At the lower salinity, the activities of GS and NADH-GDH were not altered markedly. A significant accumulation of proline was not found under either low or high salinity. The activity of Δ(1)-pyrroline-5-carboxylate reductase (P5CR) was enhanced only at 200 mM NaCl but remained unchanged at 100mM NaCl. The activity of Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) did not change under salinity-stressed. Addition of CaCl2 to the salt stressed plants not only lowered NaCl injury but also showed an elevated level of proline contents in response to the salinity treatment. In addition, both GS activity and corresponding polypeptides were expressed close to the level of control. Exogenous proline protects GS2 and the 32 kDa protein in photosystem II reaction center (D1) from H2O2-induced redox degradation in the chloroplast lysates of duckweed. The results suggest that calcium-induced proline accumulation may play an important role as a GS protectant under NaCl exposure in S. polyrhiza.

Calcium-mediated responses and glutamine synthetase expression in greater duckweed (Spirodela polyrhiza L.) under diethyl phthalate-induced stress.

This study was carried out to assess the influence of diethyl phthalate (DEP) alone or associated with calcium chloride (CaCl2) on greater duckweed plants, emphasizing the implications of calcium in amelioration of DEP-induced stress on plant growth. Greater duckweed were treated with DEP in variable concentrations, as 0, 0.25, 0.5, 1.0 and 2.0mM for 7 days, or treated with the same concentration either 2mM DEP or 2mM DEP plus 10mM CaCl2·2H2O in different duration 0-7 days. Treatment with 2mM DEP resulted in increasing proline content, protease activity, and ammonia accumulation in duckweed tissues. NADH-glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) and Δ(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2), two key enzymes in the glutamate pathway of proline synthesis, showed increase in activity with DEP treatment and positively correlated with proline accumulation. No further increase in proline accumulation was observed with addition of calcium chloride to the DEP-treated cultures. However, supplementation of Ca(2+) can mitigate the adverse effect of DEP, at least in part to decrease the DEP-induced superoxide accumulation and increase in GDH activity for ammonia assimilation in duckweed fronds. In addition, effects of calcium on mitigation of DEP injury were also observed in glutamine synthetase (GS; EC 6.3.1.2) expression. Both GS1 and GS2 polypeptide accumulation and the level of total GS activity were nearly equivalent to the control. Exogenous proline protects GS2 from DEP-modulated redox damage in the chloroplast lysates but there is no remarkable protection effects on D1 (the 32kDa protein in photosystem II reaction center) degradation. In conclusion, the glutamate pathway of proline synthesis might be involved in mitigation of DEP-induced injury, and calcium plays an important role in increasing GDH, P5CR, and GS expression.