Characterization and function of promoters of silicon transporter genes PeLsi1-1 and PeLsi1-2 from moso bamboo (Phyllostachys edulis).

KEY MESSAGE: Promoters of moso bamboo silicon transporter genes PeLsi1-1 and PeLsi1-2 contain elements in response to hormone, silicon, and abiotic stresses, and can drive the expression of PeLsi1-1 and PeLsi1-2 in transgene Arabidopsis. Low silicon 1 (Lsi1) transporters from different species have been shown to play an important role in influxing silicon from soil. In previous study, we cloned PeLsi1-1 and PeLsi1-2 from Phyllostachys edulis and verified that PeLsi1-1 and PeLsi1-2 have silicon uptake ability. Furthermore, in this study, the promoters of PeLsi1-1(1910 bp) and PeLsi1-2(1922 bp) were cloned. Deletion analysis identified the key regions of the PeLsi1-1 and PeLsi1-2 promoters in response to hormone, silicon, and abiotic stresses. RT-qPCR analysis indicated that PeLsi1-1 and PeLsi1-2 were regulated by hormones, salt stress and osmotic stress. In addition, we found that the driving activity of the PeLsi1-1 and PeLsi1-2 promoters was regulated by 2 mM K2SiO3 and PeLsi1-1-P3 ~ P4 and PeLsi1-2-P4 ~ 5 were the regions regulated by silicon. Overexpression of PeLsi1-1 or PeLsi1-2 driven by 35S promoter in Arabidopsis resulted in a threefold increase of Si accumulation, whereas transgenic plants showed deleterious symptoms and dwarf seedlings and shorter roots under 2 mM Si treatment. When the 35S promoter was replaced by PeLsi1-1 or PeLsi1-2 promoter, a similar Si absorption was achieved and the transgene plants grew normally. This study, therefore, demonstrates that the promoters of PeLsi1-1 and PeLsi1-2 are indeed effective in driving the expression of moso bamboo Lsi1 genes and leading to silicon uptake.

Prevalence and molecular characterization of Mycobacterium tuberculosis complex in cattle and humans, Maiduguri, Borno state, Nigeria: a cross-sectional study.

INTRODUCTION: Globally, the highest burden of bovine and human tuberculosis resides in Africa and Asia. Tuberculosis (TB) is the second leading single infectious killer after severe acute respiratory syndrome corona virus-2 (SARSCOV-2). Bovine TB remains a treat to wild and domesticated animals, humans and hinders international trade in endemic countries like Nigeria. We aimed at determining the prevalence of bovine and human tuberculosis, and the spoligotypes of Mycobacterium tuberculosis complex in cattle and humans in Maiduguri. METHODS: We conducted a cross sectional study on bovine and human tuberculosis in Maiduguri, Borno state. We calculated sample size using the method of Thrusfield. Lesions suggestive of TB from 160 slaughtered cattle were obtained from Maiduguri Central Abattoir. Sputum samples from humans; 82 abattoir workers and 147 suspected TB patients from hospitals/clinics were obtained. Lesions and sputum samples were cultured for the isolation of Mycobacterium spp. Positive cultures were subjected genus typing, deletion analysis and selected isolates were spoligotyped. Data was analysed using SPSS VERSION 16.0. RESULTS: Prevalence of 32.5% (52/160) was obtained in cattle. Damboa local government area (LGA), where majority of the infected animals were obtained from had 35.5% bTB prevalence. All categories analysed (breed, age, sex, body conformation and score) had P-values that were not significant (P > 0.05). Sputum culture revealed a prevalence of 3.7% (3/82) from abattoir workers and 12.2% from hospitals/clinics. A significant P-value (0.03) was obtained when positive culture from abattoir and that of hospitals/clinics were compared. Out of the 52 culture positive isolates obtained from cattle, 26 (50%) belonged to M. tuberculosis complex (MTC) and 17/26 (65.4%) were characterized as M. bovis. In humans, 7/12 (58.3%) MTC obtained were characterized as M. tuberculosis. Spoligotyping revealed SB0944 and SB1025 in cattle, while SIT838, SIT61 of LAM10_CAM and SIT1054, SIT46 of Haarlem (H) families were obtained from humans. CONCLUSIONS: Cattle in Damboa LGA need to be screened for bTB as majority of the infected animals were brought from there. Our findings revealed the presence of SB0944 and SB1025 spoligotypes from cattle in Borno state. We isolated M. tuberculosis strain of the H family mainly domiciled in Europe from humans.

GUS Reporter-Aided Promoter Deletion Analysis of A. thaliana POLYAMINE OXIDASE 3.

Polyamine oxidases (PAOs) have been correlated with numerous physiological and developmental processes, as well as responses to biotic and abiotic stress conditions. Their transcriptional regulation is driven by signals generated by various developmental and environmental cues, including phytohormones. However, the inductive mechanism(s) of the corresponding genes remains elusive. Out of the five previously characterized Arabidopsis PAO genes, none of their regulatory sequences have been analyzed to date. In this study, a GUS reporter-aided promoter deletion approach was used to investigate the transcriptional regulation of AtPAO3 during normal growth and development as well as under various inductive environments. AtPAO3 contains an upstream open reading frame (uORF) and a short inter-cistronic sequence, while the integrity of both appears to be crucial for the proper regulation of gene expression. The full-length promoter contains several cis-acting elements that regulate the tissue-specific expression of AtPAO3 during normal growth and development. Furthermore, a number of TFBS that are involved in gene induction under various abiotic stress conditions display an additive effect on gene expression. Taken together, our data indicate that the transcription of AtPAO3 is regulated by multiple environmental factors, which probably work alongside hormonal signals and shed light on the fine-tuning mechanisms of PAO regulation.

Identification of the inducible activity in the promoter of the soybean BBI-DII gene exposed to abiotic stress or abscisic acid.

The expression of the soybean Bowman-Birk proteinase isoinhibitor DII (BBI-DII) gene and the inducible activity of its promoter were studied under salt, drought, low temperature, and abscisic acid (ABA) exposure conditions. The BBI-DII gene was induced by salt, drought, low temperature, and ABA, and the relative expression levels were 103.09-, 107.01-, 17.25- and 27.24-fold, respectively, compared with the untreated control. The putative promoter, designated BP1 (- 1255 to + 872 bp), located 5'-upstream of the BBI-DII gene was cloned. The expression of the GUS gene in pCAM-BP1 transgenic tobacco plants was highest at 5 h after treatment with salt, drought, low temperature and ABA, especially under salt and drought. Using histochemical staining and fluorescence analysis of GUS, BP1 activity under salt and drought conditions after 5 h was 1.03 and 1.07-fold, respectively, compared with that of the CaMV35S promoter. Based on a 5' deletion analysis, the segment (+ 41 to + 474 bp) was the basal region that responded to salt and drought, whereas the segment (- 820 to + 41 bp) was the area that responded to increased salt and drought activity. The BP2 (- 820 to + 872) activities were 0.98- and 1.02-fold compared with that of BP1 under salt and drought conditions and was 435 bp shorter than BP1. The salt- and drought-inducible activities of the BP2 promoter in the roots, stems, and leaves of transgenic tobacco plants were stable. Taken together, BP2 is more suitable than the BP1 promoter for the study and molecular breeding of stress-resistant soybean plants.

[Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide].

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

Functional analysis of TmHKT1;4-A2 promoter through deletion analysis provides new insight into the regulatory mechanism underlying abiotic stress adaptation.

MAIN CONCLUSION: Bioinformatic, molecular, and biochemical analysis were performed to get more insight into the regulatory mechanism by which TmHKT1;4-A2 is regulated. HKT transporters from different plant species have been shown to play important role in plant response to salt. In previous work, TmHKT1;4-A2 gene from Triticum monococcum has been characterized as a major gene for Nax1 QTL (Tounsi et al. Plant Cell Physiol 57:2047-2057, 2016). So far, little is known about its regulatory mechanism. In this study, the promoter region of TmHKT1;4-A2 (1400 bp) was isolated and considered as the full-length promoter (PA2-1400). In silico analysis revealed the presence of important cis-acting elements related to abiotic stresses and phytohormones. Interestingly, our real-time RT-PCR analysis provided evidence that TmHKT1;4-A2 is regulated not only by salt stress but also by osmotic, heavy metal, oxidative, and hormones stresses. In transgenic Arabidopsis plants, TmHKT1;4-A2 is strongly active in vascular tissues of roots and leaves. Through 5'-end deletion analysis, we showed that PA2-1400 promoter is able to drive strong GUS activity under normal conditions and in response to different stresses compared to PA2-824 and PA2-366 promoters. These findings provide new information on the regulatory mechanism of TmHKT1;4-A2 and shed more light on its role under different stresses.

Cross-Talk between Overlap Interactions in Biomolecules: A Case Study of the ß-Turn Motif.

Noncovalent interactions play a pivotal role in regulating protein conformation, stability and dynamics. Among the quantum mechanical (QM) overlap-based noncovalent interactions, n→π* is the best understood with studies ranging from small molecules to ß-turns of model proteins such as GB1. However, these investigations do not explore the interplay between multiple overlap interactions in contributing to local structure and stability. In this work, we identify and characterize all noncovalent overlap interactions in the ß-turn, an important secondary structural element that facilitates the folding of a polypeptide chain. Invoking a QM framework of natural bond orbitals, we demonstrate the role of several additional interactions such as n→σ* and π→π* that are energetically comparable to or larger than n→π*. We find that these interactions are sensitive to changes in the side chain of the residues in the ß-turn of GB1, suggesting that the n→π* may not be the only component in dictating ß-turn conformation and stability. Furthermore, a database search of n→σ* and π→π* in the PDB reveals that they are prevalent in most proteins and have significant interaction energies (∼1 kcal/mol). This indicates that all overlap interactions must be taken into account to obtain a comprehensive picture of their contributions to protein structure and energetics. Lastly, based on the extent of QM overlaps and interaction energies, we propose geometric criteria using which these additional interactions can be efficiently tracked in broad database searches.

Molecular cloning and functional characterization of a seed-specific VvßVPE gene promoter from Vitis vinifera.

MAIN CONCLUSION: The grapevine VvßVPE promoter is specifically expressed in the seed. The - 1306~- 1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)ßVPE is a ß-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvßVPE promoter (pVvßVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvßVPE coding sequence is consistent. Two ßVPE promoters were constructed and transformed into Arabidopsis thaliana via ß-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvßVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvßVPE promoters and found each pVvßVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core - 1306~- 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region - 1704~- 1306 bp inhibits, and the region - 705~- 861 bp promotes gene expression of VvßVPE. Our results demonstrate that pVvßVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvßVPE (- 1306~- 1045 bp) is the key one responsible for seed-specific expression.

Characterization of wheat TaSnRK2.7 promoter in Arabidopsis.

MAIN CONCLUSION: Expression of TaSnRK2.7 promoter is strongly induced under abiotic stress and could be used as a valuable tool for improving plant stress resistance via transgenic techniques. The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family plays pivotal roles in response to abiotic stresses (drought, salinity and cold). Here, we studied the expression of five wheat TaSnRK2.7 promoter-5'-deletion constructs (- 2547, - 1621, - 806, - 599, and - 254) fused to beta-glucuronidase (GUS) in Arabidopsis. Tissue-expression analysis revealed that the - 254 to ATG fragment was sufficient for inducing GUS expression in hypocotyls. Additionally, the - 806 to - 599 and - 2547 to - 1621 fragments contained leaf- and root-specific elements, respectively. Deletion analysis showed that these fragments were unresponsive to ABA treatment, suggesting that TaSnRK2.7 participates in an ABA-independent signaling pathway. Assays examining stress responses of constructs demonstrated that the - 599 to - 254 and - 806 to - 599 fragments contained elements responsive to abiotic and osmotic stress, respectively. The TaSnRK2.7 promoter contained enhancers from - 806 to - 254 and - 2547 to - 1621, while the - 1621 to - 806 fragment contained negative regulatory elements that restrict root and leaf gene expression in response to abiotic stress. Furthermore, under drought and salt stress, the TaSnRK2.7 promoter conferred greater gene expression in leaves than the rd29A promoter, even though both were induced by abiotic stress. These findings enhance our understanding of the molecular mechanisms behind TaSnRK2.7 action, which should prove useful in transgenic studies investigating stress-induced gene expression.

GCTTCA as a novel motif for regulating mesocarp-specific expression of the oil palm (Elaeis guineensis Jacq.) stearoyl-ACP desaturase gene.

KEY MESSAGE: TAAAAT and a novel motif, GCTTCA found in the oil palm stearoyl-ACP desaturase (SAD1) promoter are involved in regulating mesocarp-specific expression. Two key fatty acid biosynthetic genes, stearoyl-ACP desaturase (SAD1), and acyl-carrier protein (ACP3) in Elaeis guineensis (oil palm) showed high level of expression during the period of oil synthesis in the mesocarp [12-19 weeks after anthesis (w.a.a.)] and kernel (12-15 w.a.a.). Both genes are expressed in spear leaves at much lower levels and the expression increased by 1.5-fold to 2.5-fold following treatments with ethylene and abscisic acid (ABA). Both SAD1 and ACP3 promoters contain phytohormone-responsive, light-responsive, abiotic factors/wounding-responsive, endosperm specificity and fruit maturation/ripening regulatory motifs. The activities of the full length and six 5' deletion fragments of the SAD1 promoter were analyzed in transiently transformed oil palm tissues by quantitative ß-glucuronidase (GUS) fluorometric assay. The highest SAD1 promoter activity was observed in the mesocarp followed by kernel and the least in the leaves. GUS activity in the D3 deletion construct (- 486 to + 108) was the highest, while the D2 (- 535 to + 108) gave the lowest suggesting the presence of negative cis-acting regulatory element(s) in the deleted - 535 to - 486 (49 bp). It was found that the 49-bp region binds to the nuclear protein extract from mesocarp but not from leaves in electrophoretic mobility shift assay (EMSA). Further fine-tuned analysis of this 49-bp region using truncated DNA led to the identification of GCTTCA as a novel motif in the SAD1 promoter. Interestingly, another known fruit ripening-related motif, LECPLEACS2 (TAAAAT) was found to be required for effective binding of the novel motif to the mesocarp nuclear protein extract.

Screening of promoters from Arthrobacter sp. CGMCC 3584 using a green fluorescent protein reporter system.

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6-hydroxyl-D-nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species.

Ras-Related Nuclear Protein Ran3B Gene Is Involved in Hormone Responses in the Embryogenic Callus of Dimocarpus longan Lour.

Ras-related guanosine triphosphate (GTP)-binding nuclear protein (Ran) GTPases function as molecular switches and regulate diverse cellular events in eukaryotes. Our previous work suggested that DlRan3B is active during longan (Dimocarpus longan Lour.) somatic embryogenesis (SE) processes. Herein, subcellular localization of DlRan3B was found to be localized in the nucleus and expression profiling of DlRan3B was performed during longan SE and after exposure to plant hormones (indoleacetic acid (IAA), gibberellin A3 (GA3), salicylic acid (SA), methyl jasmonte (MeJA), and abscisic acid (ABA)). We cloned and sequenced 1569 bp of 5'-flanking sequence of DlRan3B (GenBank: JQ279697). Bioinformatic analysis indicated that the promoter contained plant hormone-related regulatory elements. Deletion analysis and responses to hormones identified stimulative and repressive regulatory elements in the DlRan3B promoter. The key elements included those responding to auxin, gibberellin, SA, MeJA, and ABA. DlRan3B was located in the nucleus and accumulated in the late stage of longan SE. The expression of DlRan3B was significantly induced by IAA, GA3, and ABA, but suppressed by SA and MeJA. Promoter transcription was induced by IAA and GA3, but suppressed by SA. Thus, DlRan3B might participate in auxin, gibberellin, and ABA responses during longan late SE, and DlRan3B is involved in phytohormone responsiveness.

GmFNSII-controlled soybean flavone metabolism responds to abiotic stresses and regulates plant salt tolerance.

Flavones, a major group of flavonoids in most plant tissues, play multiple roles in plant-environment interactions. In our study, the expression of the two soybean flavone synthase genes, GmFNSII-1 and GmFNSII-2, was significantly increased by methyl jasmonate (MeJA), glucose, mannitol and NaCl treatment, which were also found to increase flavone aglycone accumulation in Glycine max (L.) Merrill. In the GmFNSII-1 promoter, a specific CGTCA motif in the region (-979 bp to -806 bp) involved in the MeJA response was identified. Promoter deletion analysis of GmFNSII-2 revealed the presence of osmotic-responsive (-1,143 bp to -767 bp) and glucose-repressive sequence elements (-767 bp to -475 bp), which strongly supported the hypothesis that glucose induces soybean flavone production by acting as both an osmotic factor and a sugar signaling molecule simultaneously. Silencing of the GmFNSII gene clearly reduced the production of flavone aglycones (apigenin, luteolin and 7,4'-dihydroxyflavone) in hairy roots. The GmFNSII-RNAi (RNA interference) roots that had a reduced level of flavones accompanied by more malondialdehyde and H2O2 accumulation were more sensitive to salt stress compared with those of the control, and we concluded that flavones, as antioxidants, are associated with salt tolerance.

Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus.

AmCIP is a dehydrin-like protein which involved in abiotic stress tolerance in xerophytes evergreen woody plant A. mongolicus. AmCIP could be induced in the cotyledon and radicle during cold acclimation. To further elucidate the regulation of the upstream region of the gene, we isolated and characterized the promoter of AmCIP. Herein, a 1115 bp 5'-flanking region of AmCIP genomic DNA was isolated and cloned by genome walking from A. mongolicus and the segment sequence was identified as "PrAmCIP" promoter. Analysis of the promoter sequence revealed the presences of some basic cis-acting elements, which were related to various environmental stresses and plant hormones. GUS histochemical staining of transgene tobacco showed that PrAmCIP was induced by 4℃, 55℃, NaCl, mannitol and ABA, whereas it could hardly drive GUS gene expression under normal conditions. Furthermore, we constructed three deletion fragments and genetically transformed them into Arabidopsis thaliana. GUS histochemical staining showed that the MYCATERD1 element of the CP7 fragment (-189 âˆ¼ -1) may be a key element in response to drought. In conclusion, we provide an inducible promoter, PrAmCIP, which can be applied to the development of transgenic plants for abiotic stresse tolerance.

A comprehensive list of genes required for the efficient conjugation of plasmid Rts1 was determined by systematic deletion analysis.

While conjugation-related genes have been identified in many plasmids by genome sequencing, functional analyses have not yet been performed in most cases, and a full set of conjugation genes has been identified for only a few plasmids. Rts1, a prototype IncT plasmid, is a conjugative plasmid that was originally isolated from Proteus vulgaris. Here, we conducted a systematic deletion analysis of Rts1 to fully understand its conjugation system. Through this analysis along with complementation assays, we identified 32 genes that are required for the efficient conjugation of Rts1 from Escherichia coli to E. coli. In addition, the functions of the 28 genes were determined or predicted; 21 were involved in mating-pair formation, three were involved in DNA transfer and replication, including a relaxase gene belonging to the MOBH12 family, one was involved in coupling, and three were involved in transcriptional regulation. Among the functionally well-analysed conjugation systems, most of the 28 genes showed the highest similarity to those of the SXT element, which is an integrative conjugative element of Vibrio cholerae. The Rts1 conjugation gene set included all 23 genes required for the SXT system. Two groups of plasmids with conjugation systems nearly identical or very similar to that of Rts1 were also identified.

Identification of a cis-element for long glandular trichome-specific gene expression, which is targeted by a HD-ZIP IV protein.

Glandular trichomes are epidermal outgrowths that secret a variety of secondary metabolites, which not only help plants adapt to environmental stresses but also have important commercial value in fragrances, pharmaceuticals, and pesticides. In Nicotiana tabacum, it has been confirmed that a B-type cyclin, CycB2, negatively regulates the formation of long glandular trichomes (LGTs). This study aimed to identify the upstream regulatory gene involved in LGT formation by screening LGT-specific cis-elements within the NtCycB2 promoter. Using GUS as a reporter gene, the tissue-driven ability of NtCycB2 promoter showed that NtCycB2 promoter could drive GUS expression specifically in LGTs. Function analysis of a series of successive 5' truncations and synthetic segments of the NtCycB2 promoter indicated that the 87-bp region from -1221 to -1134 of the NtCycB2 promoter was required for gene expression in LGTs, and the L1-element (5'-AAAATTAATAAGAG-3') located in the 87-bp region contributed to the gene expression in the stalk of LGTs. Further Y1H and LUC assays confirmed that this L1-element exclusively binds to a HD-Zip IV protein, NtHD13. Gene function analysis revealed that NtHD13 positively controlled LGT formation, as overexpression of NtHD13 resulted in a high number of LGTs, whereas knockout of NtHD13 led to a decrease in LGTs. These findings demonstrate that NtHD13 can bind to an L1-element within the NtCycB2 promoter to regulate LGT formation.

Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

Functional Characterization of TkSRPP Promoter in Response to Hormones and Wounding Stress in Transgenic Tobacco.

Taraxacum kok-saghyz is a model species for studying natural rubber biosynthesis because its root can produce high-quality rubber. Small rubber particle protein (SRPP), a stress-related gene to multiple stress responses, involves in natural rubber biosynthesis. To investigate the transcriptional regulation of the TkSRPP promoter, the full-length promoter PR0 (2188 bp) and its four deletion derivatives, PR1 (1592 bp), PR2 (1274 bp), PR3 (934 bp), and PR4 (450 bp), were fused to ß-glucuronidase (GUS) reporter gene and transformed into tobacco. The GUS tissue staining showed that the five promoters distinctly regulated GUS expression utilizing transient transformation of tobacco. The GUS activity driven by a PR0 promoter was detected in transgenic tobacco leaves, stem and roots, suggesting that the TkSRPP promoter was not tissue-specific. Deletion analyses in transgenic tobacco have demonstrated that the PR3 from -934 bp to -450 bp core region responded strongly to the hormones, methyl jasmonate (MeJA), abscisic acid (ABA), and salicylic acid (SA), and also to injury induction. The TkSRPP gene was highly expressed under hormones and wound-induced conditions. This study reveals the regulation pattern of the SRPP promoter, and provides valuable information for studying natural rubber biosynthesis under hormones and wounding stress.

Molecular epidemiology of Mycobacterium bovis in central parts of Malawi.

Bovine tuberculosis (bTB) is a neglected disease that affects cattle and humans. The burden of bTB is higher in developing countries as compared to industrialized countries. The reasons behind this discrepancy include the fact that bTB control measures, such as testing and slaughter of infected cattle and pasteurization of milk, are not usually practised in developing countries largely because of their high cost. To improve our understanding of bTB in developing countries, molecular typing studies are essential, in particular in terms of transmission dynamics, infection sources and knowledge of circulating strains of the principal causative agent, Mycobacterium bovis. In this study, we applied a suite of molecular typing techniques encompassing deletion analysis, spoligotyping and MIRU-VNTR to isolates recovered from samples collected during the routine post-mortem of cattle at the cold storage abattoir in Lilongwe, Malawi. Out of 63 isolates, 51 (81%) belonged to the European 1. M. bovis clonal complex. Spoligotyping identified 8 profiles, with SB0131 being the predominant type (56% of isolates). Spoligotypes SB0273 and SB0425 were identified in 14% and 13%, respectively, of the isolates. MIRU-VNTR showed a high discriminatory power of 0.959 and differentiated the 8 spoligotypes to 31 genotypes. The high diversity of M. bovis within the study area suggests the infection has been circulating in the area for a considerable period of time, likely facilitated by the lack of effective control measures. We also observed genetic similarities between isolates from Malawi (this study) to isolates described in previous studies in Zambia and Mozambique, suggesting transmission links in this region. The information provided by this study provides much needed evidence for the formulation of improved bTB control strategies.

Mutational Analysis of a Highly Conserved PLSSMXP Sequence in the Small Subunit of Bacillus licheniformis γ-Glutamyltranspeptidase.

A highly conserved 458PLSSMXP464 sequence in the small subunit (S-subunit) of an industrially important Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of BlGGT clearly reveal that this peptide sequence is in close spatial proximity to the self-processing and catalytic sites of the enzyme. To probe the role of this conserved sequence, ten mutant enzymes of BlGGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of BlGGT to undergo autocatalytic processing was detrimentally affected by the deletion-associated mutations. However, loss of self-activating capacity was not obviously observed in most of the Ala-replacement mutants. The Ala-replacement mutants had a specific activity comparable to or greater than that of the wild-type enzyme; conversely, all deletion mutants completely lost their enzymatic activity. As compared with BlGGT, S460A and S461S showed greatly enhanced kcat/Km values by 2.73- and 2.67-fold, respectively. The intrinsic tryptophan fluorescence and circular dichroism spectral profiles of Ala-replacement and deletion mutants were typically similar to those of BlGGT. However, heat and guanidine hydrochloride-induced unfolding transitions of the deletion-associated mutant proteins were severely reduced as compared with the wild-type enzyme. The predictive mutant models suggest that the microenvironments required for both self-activation and catalytic reaction of BlGGT can be altered upon mutations.