VIRULENCE GENE PROFILING AND PATHOGENICITY OF Streptococcus agalactiae ISOLATED FROM TILAPIA, Oreochromis niloticus FARMS IN INDONESIA

Streptococcosis caused by Streptococcus agalactiae has become a major disease problem in tilapia culture in Indonesia. This study aimed to detect virulence genes of S. agalactiae during streptococcosis disease outbreaks in several tilapia farms in Indonesia and evaluate the correlation between biotype and virulence genes to bacterial virulence. The presence of virulence genes was determined in 10 strains of S. agalactiae isolated from farm-raised tilapia. Polymerase chain reaction (PCR) protocol was used to determine genes for cylE, hylB, lmb, bib A, PI-2b, fbs A, fbs B, gap, PI-1, and cfb in the template DNA. Pathogenicity test was carried out by intraperitoneal injection of 24 hour-cultured S. agalactiae to tilapia with 108 CFU/fish. Four isolates have seven of virulence genes (cylE, hylB, bibA, PI-2b, fbs A, fbs B, and gap genes), three isolates have six virulence genes (hylB, bib A, fbs A, fbs B, gap, cfb genes), one isolate has four virulence gene (hyl B, bib A, fbs, and cfb genes), and one isolate has one virulence gene (PI-2b gene). None of the isolates has lmb or PI-1 genes. Bacteria with more virulence genes showed higher pathogenicity post injection. Mortality of tilapia injected with b-hemolytic bacteria was 100% within the period of 14-19 hours, while non-hemolytic bacteria was 53.3%-86.6% on 14 days post-injection. Pathological changes associated with the infection by either isolate included melanosis, slow response, anorexia, ocular opacity, gasping, erratic, C-shape, and whirling. It can be concluded that S. agalactiae with more virulence genes show a higher level of pathogenicity. The presence of a virulent gene has the potential to be used as a basis for selecting candidate isolates and designing vaccine compositions as an effort to prevent streptococcosis infection in tilapia in Indonesia.

Gap genes are involved in inviability in hybrids between Drosophila melanogaster and D. santomea

ABSTRACTEvolved changes within species lead to the inevitable loss of viability in hybrids. Inviability is also a convenient phenotype to genetically map and validate functionally divergent genes and pathways differentiating closely related species. Here we identify the Drosophila melanogaster form of the highly conserved essential gap gene giant (gt) as a key genetic determinant of hybrid inviability in crosses with D. santomea. We show that the coding region of this allele in D. melanogaster/D. santomea hybrids is sufficient to cause embryonic inviability not seen in either pure species. Further genetic analysis indicates that tailless (tll), another gap gene, is also involved in the hybrid defects. giant and tll are both members of the gap gene network of transcription factors that participate in establishing anterior-posterior specification of the dipteran embryo, a highly conserved developmental process. Genes whose outputs in this process are functionally conserved nevertheless evolve over short timescales to cause inviability in hybrids.

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal quantitative regulatory dynamics from a complex genetic network in vivo. We engineer light-controlled variants of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent expression of giant and hunchback and delayed repression of Kruppel. In contrast, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a previously unreported role for Bicoid in suppressing knirps expression. Acute modulation of transcription factor concentration while simultaneously recording output gene activity represents a powerful approach for studying how gene circuit elements are coupled to cell identification and complex body pattern formation in vivo.

FunOrder: A robust and semi-automated method for the identification of essential biosynthetic genes through computational molecular co-evolution

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.

The neuroblast timer gene nubbin exhibits functional redundancy with gap genes to regulate segment identity in Tribolium

The neuroblast timer genes hunchback, Krüppel, nubbin, and castor are expressed in temporal sequence in neural stem cells, and in corresponding spatial sequence along the Drosophila blastoderm. As canonical gap genes, hunchback and Krüppel play a crucial role in insect segmentation, but the roles of nubbin and castor in this process remain ambiguous. We have investigated the expression and functions of nubbin and castor during segmentation in the beetle Tribolium. We show that Tc-hunchback, Tc-Krüppel, Tc-nubbin and Tc-castor are expressed sequentially in the segment addition zone, and that Tc-nubbin regulates segment identity redundantly with two previously described gap/gap-like genes, Tc-giant and Tc-knirps. Simultaneous knockdown of Tc-nubbin, Tc-giant and Tc-knirps results in the formation of ectopic legs on abdominal segments. This homeotic transformation is caused by loss of abdominal Hox gene expression, likely due to expanded Tc-Krüppel expression. Our findings support the theory that the neuroblast timer series was co-opted for use in insect segment patterning, and contribute to our growing understanding of the evolution and function of the gap gene network outside of Drosophila.

Annotation of segmentation pathway genes in the Asian citrus psyllid, Diaphorina citri

Insects have a segmented body plan that is established during embryogenesis when the anterior–posterior (A–P) axis is divided into repeated units by a cascade of gene expression. The cascade is initiated by protein gradients created by translation of maternally provided mRNAs, localized at the anterior and posterior poles of the embryo. Combinations of these proteins activate specific gap genes to divide the embryo into distinct regions along the anterior–posterior axis. Gap genes then activate pair-rule genes, which are usually expressed in parts of every other segment. The pair-rule genes, in turn, activate expression of segment polarity genes in a portion of each segment. The segmentation genes are generally conserved among insects, although there is considerable variation in how they are deployed. We annotated 25 segmentation gene homologs in the Asian citrus psyllid, Diaphorina citri. Most of the genes expected to be present in D. citri based on their phylogenetic distribution in other insects were identified and annotated. Two exceptions were eagle and invected, which are present in at least some hemipterans, but were not found in D. citri. Many of the segmentation pathway genes are likely to be essential for D. citri development, and thus they may be useful targets for gene-based pest control methods.

The neuroblast timer gene nubbin exhibits functional redundancy with gap genes to regulate segment identity in Tribolium

In Drosophila, segmentation genes of the gap class form a regulatory network that positions segment boundaries and assigns segment identities. This gene network shows striking parallels with another gene network known as the neuroblast timer series. The neuroblast timer genes hunchback, Krüppel, nubbin, and castor are expressed in temporal sequence in neural stem cells to regulate the fate of their progeny. These same four genes are expressed in corresponding spatial sequence along the Drosophila blastoderm. The first two, hunchback and Krüppel, are canonical gap genes, but nubbin and castor have limited or no roles in Drosophila segmentation. Whether nubbin and castor regulate segmentation in insects with the ancestral, sequential mode of segmentation remains largely unexplored. We have investigated the expression and functions of nubbin and castor during segment patterning in the sequentially-segmenting beetle Tribolium. Using multiplex fluorescent in situ hybridisation, we show that Tc-hunchback, Tc-Krüppel, Tc-nubbin and Tc-castor are expressed sequentially in the segment addition zone of Tribolium, in the same order as they are expressed in Drosophila neuroblasts. Furthermore, simultaneous disruption of multiple genes reveals that Tc-nubbin regulates segment identity, but does so redundantly with two previously described gap/gap-like genes, Tc-giant and Tc-knirps. Knockdown of two or more of these genes results in the formation of up to seven pairs of ectopic legs on abdominal segments. We show that this homeotic transformation is caused by loss of abdominal Hox gene expression, likely due to expanded Tc-Krüppel expression. Our findings support the theory that the neuroblast timer series was co-opted for use in insect segment patterning, and contribute to our growing understanding of the evolution and function of the gap gene network outside of Drosophila.

The Functional Order (FunOrder) tool – Identification of essential biosynthetic genes through computational molecular co-evolution

ABSTRACTSecondary metabolites (SMs) are a vast group of compounds with different structures and properties. Humankind uses SMs as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs but they have problems with distinguishing essential genes from gap genes and defining the borders of a BGC. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or co-expression analyses. In this study, we developed a novel tool that allows automated identification of essential genes in a BGC based solely on genomic data. The Functional Order (FunOrder) tool – Identification of essential biosynthetic genes through computational molecular co-evolution – searches for co-evolutionary linked genes in the BGCs. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs, including antibiotics and other pharmaceuticals.

Segmentation pathway genes in the Asian citrus psyllid, Diaphorina citri

Insects have a segmented body plan that is established during embryogenesis when the anterior-posterior (A-P) axis is divided into repeated units by a cascade of gene expression. The cascade is initiated by protein gradients created by translation of maternally provided mRNAs, localized at the anterior and posterior poles of the embryo. Particular combinations of these proteins activate specific gap genes to divide the embryo into distinct regions along the A-P axis. Gap genes then activate pair-rule genes, which are usually expressed in part of every other segment. The pair-rule genes, in turn, activate expression of segment polarity genes in a portion of each segment. The segmentation genes are generally conserved among insects, although there is considerable variation in how they are deployed. We annotated 24 segmentation gene homologs in the Asian citrus psyllid, Diaphorina citri. We identified most of the genes that were expected to be present based on known phylogenetic distribution. Two exceptions were eagle and invected, which are present in at least some hemipterans, but were not identified in D. citri. Many of these genes are likely to be essential for D. citri development and thus may be useful targets for pest control methods.