A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants

Morales, Víctor; Bäckman, Assar; Bagdasarian, Michael

doi:10.1016/0378-1119(91)90007-x

Cited by 468 publications

(208 citation statements)

References 24 publications

Supporting

Mentioning

206

Contrasting

Order By: Relevance

“…Correct integration of pKNG101-ybeY into the ybeY gene, which generated strain YeO3-DybeY, was verified by PCR using different combinations of following primers: ybeY-F, ybeY-R, ybeY-outR (59-GGGCGTTATTATACCCGTCA-39), ybeYoutF (59-GCGTGGTCATTGCTTATGAA-39), pKNG-F (59-GGAAA-GGACCCGTAAAGTGA-39) and pKNG-R (59-CGATACACTTCCG-CTCAGGT-39). To construct a plasmid carrying the WT ybeY gene for complementation experiments the full ybeY gene was PCR amplified using primers ybeY-outF and ybeY-outR and the fragment was ligated into SmaI-digested and SAP-treated expression vector pMMB207 (Morales et al, 1991). The correct orientation of the ybeY gene in the obtained plasmid pMMB207-ybeY was verified by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3

2015

View full text Add to dashboard Cite

YbeY was recently recognized as an endoribonuclease playing a role in ribosome biosynthesis. In Escherichia coli it functions as a single-strand-specific RNase that processes the 39 end of the 16S rRNA and is crucial for the late-stage 70S ribosome quality control system. Here we report that YbeY is not essential in Yersinia enterocolitica serotype O:3, yet its absence strongly compromised the bacterium. The lack of YbeY resulted in misprocessing of 16S rRNA and a severe decrease of growth rate with complete growth arrest observed at elevated temperatures. Moreover, a ybeY mutation severely disturbed regulation of the Yersinia virulence plasmid (pYV) genes and affected the expression of regulatory small RNA species. Transcription of the pYV genes was upregulated in the ybeY mutant at 22 6C; the same genes were repressed in the wildtype bacterium. Furthermore, ybeY inactivation impaired many virulence-related features, such as resistance to elevated temperature and acid, and hindered utilization of different carbohydrates. In addition, the ybeY mutant strain showed decreased infectivity in a tissue culture infection model, especially at the stage of cell adhesion. Taken together, this study demonstrates the crucial role of YbeY in Y. enterocolitica O:3 physiology and pathogenicity.

show abstract

Section: Methodsmentioning

confidence: 99%

Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3

2015

View full text Add to dashboard Cite

show abstract

“…To construct a plasmid carrying the WT rfaH gene for complementation experiments, the full rfaH gene with its promoter region was amplified by PCR using primers P-rfaH-F and C-rfaH-R (Table S1), and the fragment was ligated into Eco RV-digested and SAPtreated expression vector pTM100 (Michiels & Cornelis, 1991;Morales et al, 1991) to obtain plasmid pTM100-rfaH (Fig. S1).…”

Section: Methodsmentioning

confidence: 99%

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Leskinen

Varjosalo

et al. 2015

Microbiology

View full text Add to dashboard Cite

The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA sequencing of the WT and DrfaH strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the DrfaH strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the DrfaH strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the DrfaH strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the DrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in Y. enterocolitica O:3. It may be speculated that RfaH might have an in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.

show abstract

“…Cloning and Overexpression of VC1649, VC1200, and VCA0803 Loci-The VC1649 and VCA0803 loci were amplified with the following primers pairs: Fwd, 5Ј-GAGCTCGGG-AGTTATCAGAGGTATCT-3Ј, and Rev, 5Ј-GCATGCAAAT-TGGCTATCGATAGATC-3Ј; Fwd, 5Ј-TCTAGATTCAACC-AATGAGGTGACGC-3Ј, and Rev 5Ј-AAGCTTTCTCAA-ATCAAACACACCTTGATC-3Ј, respectively, and subsequently cloned into SacI-SphI-and XbaI-HindIII-digested pMMB67EH (49) to create pVesC and pVesA, respectively. To overproduce protein encoded by the VC1200 loci, plasmid pVesB was used (50).…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Sikora¹,

Zielke²,

Lawrence

et al. 2011

Journal of Biological Chemistry

106

143

View full text Add to dashboard Cite

The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many Gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961.Gram-negative bacteria have evolved at least six secretion pathways devoted to the transport of proteins through the cell envelope into either the extracellular environment or directly into host cells (1, 2). The type II secretion (T2S) 2 system was first discovered in Klebsiella oxytoca and has been shown to be widely distributed among ␥-proteobacteria (3-6). Depending on the bacterial species, the T2S complex consists of 12-16 different constituents that form a multiprotein apparatus spanning the entire cell envelope (7,8). The conserved components of the T2S machinery include the cytoplasmic ATPase (T2S E), the inner membrane platform (T2S C, F, L, and M), a pilus-like structure (T2S G-K), a protein responsible for the processing of pseudopilins (T2S O), and the secretion pore (T2S D) embedded in the outer membrane (9). The exoprotein precursors are synthesized with N-terminal signal peptides that direct them into the periplasmic space via either the Sec or Tat transport systems (10, 11). After obtaining tertiary conformation, the exoproteins enter the T2S machinery and are subsequently translocated into the extracellular milieu (12, 13). Many key steps in the secretion process are still not well understood, including how the exoproteins are recognized by the T2S system, and a specific secretion signal common to known substrates has not yet been identified....

show abstract

A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants

Cited by 468 publications

References 24 publications

Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3

Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Contact Info

Product

Resources

About