Modular Cloning of the Type III Secretion Gene Cluster from the Plant-Pathogenic BacteriumXanthomonas euvesicatoria

2019 ◽  
Vol 8 (3) ◽  
pp. 532-547 ◽  
Author(s):  
Jens Hausner ◽  
Michael Jordan ◽  
Christian Otten ◽  
Sylvestre Marillonnet ◽  
Daniela Büttner
Keyword(s):  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii ◽  
Modular Cloning

scholarly journals A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis

2005 ◽  
Vol 187 (23) ◽  
pp. 8164-8171 ◽  
Author(s):  
Diana Ideses ◽  
Uri Gophna ◽  
Yossi Paitan ◽  
Roy R. Chaudhuri ◽  
Mark J. Pallen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Secretion System ◽  
Effector Proteins ◽  
E Coli ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2sepsis, which, although degenerate, contributes to pathogenesis. ETT2sepsis has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.

scholarly journals Temperature-Dependent Expression of Type III Secretion System Genes and Its Regulation in Bradyrhizobium japonicum

2010 ◽  
Vol 23 (5) ◽  
pp. 628-637 ◽  
Author(s):  
Min Wei ◽  
Keisuke Takeshima ◽  
Tadashi Yokoyama ◽  
Kiwamu Minamisawa ◽  
Hisayuki Mitsui ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Type Iii Secretion ◽  
Bradyrhizobium Japonicum ◽  
Expression Profiles ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Large Gene ◽  
Efficient Induction ◽  
Glycine Max L

The genome-wide expression profiles of Bradyrhizobium japonicum in response to soybean (Glycine max (L.) Merr.) seed extract (SSE) and genistein were monitored with time at a low temperature (15°C). A comparison with the expression profiles of the B. japonicum genome previously captured at the common growth temperature (30°C) revealed that the expression of SSE preferentially induced genomic loci, including a large gene cluster encoding the type III secretion system (T3SS), were considerably delayed at 15°C, whereas most nodulation (nod) gene loci, including nodD1 and nodW, were rapidly and strongly induced by both SSE and genistein. Induction of the T3SS genes was progressively activated upon the elevation of temperature to 30°C and positively responded to culture population density. In addition, genes nolA and nodD2 were dramatically induced by SSE, concomitantly with the expression of T3SS genes. However, the deletion mutation of nodD2 but not nolA led to elimination of the T3SS genes expression. These results indicate that the expression of the T3SS gene cluster is tightly regulated with integration of environmental cues such as temperature and that NodD2 may be involved in its efficient induction in B. japonicum.

scholarly journals The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from Escherichia coli, Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition

2004 ◽  
Vol 186 (11) ◽  
pp. 3547-3560 ◽  
Author(s):  
Chuan-Peng Ren ◽  
Roy R. Chaudhuri ◽  
Amanda Fivian ◽  
Christopher M. Bailey ◽  
Martin Antonio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Genome Sequences ◽  
Phylogenetic Structure ◽  
E Coli ◽  
Type Iii ◽  
K 12

ABSTRACT ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.

scholarly journals The Flag-2 Locus, an Ancestral Gene Cluster, Is Potentially Associated with a Novel Flagellar System from Escherichia coli

2005 ◽  
Vol 187 (4) ◽  
pp. 1430-1440 ◽  
Author(s):  
Chuan-Peng Ren ◽  
Scott A. Beatson ◽  
Julian Parkhill ◽  
Mark J. Pallen
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Close Relative ◽  
Secretion Systems ◽  
Ancestral Gene ◽  
E Coli ◽  
Type Iii ◽  
K 12

ABSTRACT Escherichia coli K-12 possesses two adjacent, divergent, promoterless flagellar genes, fhiA-mbhA, that are absent from Salmonella enterica. Through bioinformatics analysis, we found that these genes are remnants of an ancestral 44-gene cluster and are capable of encoding a novel flagellar system, Flag-2. In enteroaggregative E. coli strain 042, there is a frameshift in lfgC that is likely to have inactivated the system in this strain. Tiling path PCR studies showed that the Flag-2 cluster is present in 15 of 72 of the well-characterized ECOR strains. The Flag-2 system resembles the lateral flagellar systems of Aeromonas and Vibrio, particularly in its apparent dependence on RpoN. Unlike the conventional Flag-1 flagellin, the Flag-2 flagellin shows a remarkable lack of sequence polymorphism. The Flag-2 gene cluster encodes a flagellar type III secretion system (including a dedicated flagellar sigma-antisigma combination), thus raising the number of distinct type III secretion systems in Escherichia/Shigella to five. The presence of the Flag-2 cluster at identical sites in E. coli and its close relative Citrobacter rodentium, combined with its absence from S. enterica, suggests that it was acquired by horizontal gene transfer after the former two species diverged from Salmonella. The presence of Flag-2-like gene clusters in Yersinia pestis, Yersinia pseudotuberculosis, and Chromobacterium violaceum suggests that coexistence of two flagellar systems within the same species is more common than previously suspected. The fact that the Flag-2 gene cluster was not discovered in the first 10 Escherichia/Shigella genome sequences studied emphasizes the importance of maintaining an energetic program of genome sequencing for this important taxonomic group.

scholarly journals Type III Secretion Contributes to the Pathogenesis of the Soft-Rot Pathogen Erwinia carotovora: Partial Characterization of the hrp Gene Cluster

2001 ◽  
Vol 14 (8) ◽  
pp. 962-968 ◽  
Author(s):  
A. Rantakari ◽  
O. Virtaharju ◽  
S. Vähämiko ◽  
S. Taira ◽  
E. T. Palva ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Type Iii Secretion ◽  
Plant Pathogens ◽  
Erwinia Carotovora ◽  
Soft Rot ◽  
Hypersensitive Reaction ◽  
Virulence Determinants ◽  
Type Iii ◽  
Hrp Genes ◽  
Soft Rot Erwinia

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative σL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC¯ strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.

scholarly journals The Erwinia chrysanthemi EC16 hrp/hrc Gene Cluster Encodes an Active Hrp Type III Secretion System That Is Flanked by Virulence Genes Functionally Unrelated to the Hrp System

2004 ◽  
Vol 17 (6) ◽  
pp. 644-653 ◽  
Author(s):  
Clemencia M. Rojas ◽  
Jong Hyun Ham ◽  
Lisa M. Schechter ◽  
Jihyun F. Kim ◽  
Steven V. Beer ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Virulence Factors ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Amino Acid Identity ◽  
Secretion System ◽  
Erwinia Chrysanthemi ◽  
Type Iii

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana However, VirA(1–61)¯ Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrp/hrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.

scholarly journals Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains

2012 ◽  
Vol 12 (1) ◽  
pp. 188 ◽  
Author(s):  
Anastasia D Gazi ◽  
Panagiotis F Sarris ◽  
Vasiliki E Fadouloglou ◽  
Spyridoula N Charova ◽  
Nikolaos Mathioudakis ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii

scholarly journals Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

2014 ◽  
Vol 80 (19) ◽  
pp. 5927-5934 ◽  
Author(s):  
Kevin J. Metcalf ◽  
Casey Finnerty ◽  
Anum Azam ◽  
Elias Valdivia ◽  
Danielle Tullman-Ercek
Keyword(s):  
Gene Cluster ◽  
Type Iii Secretion ◽  
Transcriptional Control ◽  
Culture Media ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Secreted Protein ◽  
Heterologous Proteins ◽  
Content Type ◽  
Type Iii

ABSTRACTThe type III secretion system (T3SS) encoded at theSalmonellapathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS ofSalmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression ofhilAincreases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer fromhilAoverexpression also correlates to increased enzyme activity in the culture supernatant.

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