Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity

Marits, Reet; Kõiv, Viia; Laasik, Eve; Mäe, Andres

doi:10.1099/13500872-145-8-1959

Cited by 65 publications

(49 citation statements)

References 34 publications

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“…Pectinolytic activity alone was not sufficient to enable a bacterial strain to spoil bean sprouts, but spoilage required the simultaneous expression of proteolytic and pectinolytic activities. Marits et al (45) also found that protease activity was necessary for the normal progression of the disease symptoms caused by E. carotovora subsp. carotovora in living plants.…”

Section: Discussionmentioning

confidence: 99%

Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts

Rasch

Andersen

Nielsen

et al. 2005

Appl Environ Microbiol

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Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10 8 to 10 9 CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of N-3-oxohexanoyl-L-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (I gene) from a spoilage Pectobacterium was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in Erwinia carotovora. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in Enterobacteriaceae. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensingregulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.Many gram-negative bacteria use chemical signal molecules, acylated homoserine lactones (AHLs), to regulate the expression of particular phenotypes as a function of cell density. This phenomenon, known as quorum sensing (QS), is involved in the expression of virulence factors in several pathogenic bacteria and in the expression of factors important for the bacterial colonization of higher organisms (35,52,59,62,63). The growth and activity of gram-negative bacteria are also important in food microbiology, where spoilage of fresh foods, such as fish, meat, milk, and vegetables, is mostly a consequence of the degradative activity of gram-negative bacteria growing to high densities (10 8 to 10 9 CFU per gram). Food spoilage is a major economic and societal problem, and large amounts of foods are lost due to bacterial growth and spoilage (9). Understanding of the microbial processes leading to spoilage would facilitate development of novel preservation techniques and reduce loss of food. We hypothesized that bacterial fooddegradative processes could be regulated by QS, and if QS systems...

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Section: Discussionmentioning

confidence: 99%

Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts

Rasch

Andersen

Nielsen

et al. 2005

Appl Environ Microbiol

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“…Metalloproteases secreted through the Prt T1SS contribute to SRE virulence (59). In Pectobacterium, the T1SS is upregulated by plant extracts and acylhomoserine lactone (AHL), and controlled by GacAS (97,98). In Dickeya, this system is controlled by PecS (59,104) cand GacAS (85).…”

Section: Type I Secretion System: Metalloproteases and Adhesinsmentioning

confidence: 99%

The Role of Secretion Systems and Small Molecules in Soft-Rot Enterobacteriaceae Pathogenicity

Charkowski¹,

Blanco

Condemine

et al. 2012

Annu. Rev. Phytopathol.

220

251

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Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

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“…carotovorum (Pcc) is largely due its capacity to synthesize and secrete plant cell wall degrading enzymes (PCWDE), including pectinases, cellulases and protease Marits et al, 1999;Mäe et al, 1995;Pirhonen et al, 1991). Survival of infecting Pcc largely depends on production of PCWDE for the transition from latent infection to disease state.…”

Section: Introductionmentioning

confidence: 99%

A role for the Rcs phosphorelay in regulating expression of plant cell wall degrading enzymes in Pectobacterium carotovorum subsp. carotovorum

et al. 2010

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The Rcs phosphorelay is a signal transduction system that influences the virulence phenotype of several pathogenic bacteria. In the plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) the response regulator of the Rcs phosphorelay, RcsB, represses expression of plant cell wall degrading enzymes (PCWDE) and motility. The focus of this study was to identify genes directly regulated by the binding of RcsB that also regulate expression of PCWDE genes in Pcc. RcsB-binding sites within the regulatory regions of the flhDC operon and the rprA and rsmB genes were identified using DNase I protection assays, while in vivo studies using flhDC : : gusA, rsmB : : gusA and rprA : : gusA gene fusions revealed gene regulation. These experiments demonstrated that the operon flhDC, a flagellar master regulator, was repressed by RcsB, and transcription of rprA was activated by RcsB. Regulation of the rsmB promoter by RcsB is more complicated. Our results show that RcsB represses rsmB expression mainly through modulating flhDC transcription. Neverthless, direct binding of RcsB on the rsmB promoter region is possible in certain conditions. Using an rprA-negative mutant, it was further demonstrated that RprA RNA is not essential for regulating expression of PCWDE under the conditions tested, whereas overexpression of rprA increased protease expression in wild-type cells. Stationary-phase sigma factor, RpoS, is the only known target gene for RprA RNA in Escherichia coli; however, in Pcc the effect of RprA RNA was found to be rpoS-independent. Overall, our results show that the Rcs phosphorelay negatively affects expression of PCWDE by inhibiting expression of flhDC and rsmB. INTRODUCTIONThe phytopathogenicity of Pectobacterium carotovorum subsp. carotovorum (Pcc) is largely due its capacity to synthesize and secrete plant cell wall degrading enzymes (PCWDE), including pectinases, cellulases and protease Marits et al., 1999; Mäe et al., 1995;Pirhonen et al., 1991). Survival of infecting Pcc largely depends on production of PCWDE for the transition from latent infection to disease state. A number of global and specific regulatory genes contribute to expression of PCWDE in Pcc (Burr et al., 2006;Chatterjee et al., 1995;Cui et al., 1995Cui et al., , 1999Cui et al., , 2001Cui et al., , 2005Eriksson et al., 1998;Flego et al., 2000;Harris et al., 1998; Hyytiäinen et al., 2003;Liu et al., 1998Liu et al., , 1999 Sjöblom et al., 2006). The posttranscriptional Rsm system plays a key role in regulating the expression of PCWDE Cui et al., 1995;Liu et al., 1998).The Rsm system consists of two regulators in Pcc: a small protein, RsmA, and RsmB RNA. RsmA is a post-transcriptional global regulator that is thought to bind mRNAs of PCWDE genes and affect their stability, whereas RsmB RNA binds multiple molecules of RsmA, thereby neutralizing its effect (Liu et al., 1998). Recently, the Rsm system was shown to be dependent on expression of a global regulator of flagellar genes, FlhDC. FlhDC activates the expression of PCWDE in Pcc strain 7...

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Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity

Cited by 65 publications

References 34 publications

Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts

Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts

The Role of Secretion Systems and Small Molecules in Soft-Rot Enterobacteriaceae Pathogenicity

A role for the Rcs phosphorelay in regulating expression of plant cell wall degrading enzymes in Pectobacterium carotovorum subsp. carotovorum

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