Targeting of Flavobacterium Johnsoniae Proteins for Secretion By the Type IX Secretion System

Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to family TIGR04183 (type A CTDs). This thesis focuses on the functional characterization of diverse CTDs for secretion by the F. johnsoniae T9SS. Fusion of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign protein sfGFP that had a signal peptide at the amino terminus (SP-sfGFP) resulted in secretion across the outer membrane. In each case approximately 80 to 100 amino acids from the extreme carboxy-terminus was needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting secretion of sfGFP by the F. johnsoniae T9SS. The F. johnsoniae adhesin SprB is propelled rapidly along the cell surface resulting in gliding motility. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to family TIGR04131, which we refer to as a type B CTD. Type B CTDs are common in the Bacteroidetes but little is known regarding their roles in secretion. The secretion of the foreign protein sfGFP fused to an N-terminal SP and to C-terminal regions of SprB (SP-sfGFP-CTDSprB) was analyzed. CTDs of 218 AAs or longer resulted in secretion whereas a CTD of 149 AAs did not. sprF, which lies downstream of sprB, is known to be required for SprB secretion. SP-sfGFP-CTDSprB also required SprF for secretion. Efficient secretion only occurred when SP-sfGFP-CTDSprB and SprF were expressed together. Under these conditions CTDs of 218 AAs and 448 AAs resulted in secretion of soluble sfGFP, whereas longer CTDs (663 and 1182 AAs) resulted in attachment of sfGFP to the cell surface. Most F. johnsoniae genes encoding proteins with type B CTDs lie immediately upstream of sprF-like genes. The CTD from one such protein, Fjoh_3952, facilitated secretion of sfGFP only when it was coexpressed with its cognate SprF-like protein, Fjoh_3951. Secretion did not occur when SP-sfGFP-CTDFjoh_3952 was expressed with SprF, or when SP-sfGFP-CTDSprB was expressed with Fjoh_3951. The results highlight the need for extended regions of type B CTDs for secretion and cell-surface localization, and the requirement for the appropriate SprF-like protein for secretion. Since type B CTD-containing proteins and associated SprF-like proteins are common among members of the phylum Bacteroidetes the unique features required for secretion of these proteins may have broad implications

A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus.

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine

In Silico Prediction of Evolutionarily Conserved GC-Rich Elements Associated with Antigenic Proteins of Plasmodium falciparum

The Plasmodium falciparum genome being AT-rich, the presence of GC-rich regions suggests functional significance. Evolution imposes selection pressure to retain functionally important coding and regulatory elements. Hence searching for evolutionarily conserved GC-rich, intergenic regions in an AT-rich genome will help in discovering new coding regions and regulatory elements. We have used elevated GC content in intergenic regions coupled with sequence conservation against P. reichenowi, which is evolutionarily closely related to P. falciparum to identify potential sequences of functional importance. Interestingly, ~30% of the GC-rich, conserved sequences were associated with antigenic proteins encoded by var and rifin genes. The majority of sequences identified in the 5′ UTR of var genes are represented by short expressed sequence tags (ESTs) in cDNA libraries signifying that they are transcribed in the parasite. Additionally, 19 sequences were located in the 3′ UTR of rifins and 4 also have overlapping ESTs. Further analysis showed that several sequences associated with var genes have the capacity to encode small peptides. A previous report has shown that upstream peptides can regulate the expression of var genes hence we propose that these conserved GC-rich sequences may play roles in regulation of gene expression

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare

Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare. The F. columnare Delta gldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The Delta gldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare Delta porV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from Delta gldN and Delta porV mutants did not, suggesting that soluble toxins are secreted by the T9SS. IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.</p