Summary• Type II secretion (T2S) systems of many plant-pathogenic bacteria often secrete cell wall-degrading enzymes into the plant apoplast.• Here, we show that the Xps-T2S system from the plant pathogen Xanthomonas campestris pv vesicatoria (Xcv) promotes disease and contributes to the translocation of effector proteins that are delivered into the plant cell by the type III secretion (T3S) system.• The Xcs-T2S system instead lacks an obvious virulence function. However, individual xcs genes can partially complement mutants in homologous xps genes, indicating that they encode functional components of T2S systems. Enzyme activity assays showed that the Xps system contributes to secretion of proteases and xylanases. We identified the virulence-associated xylanase XynC as a substrate of the Xps system. However, homologs of known T2S substrates from other Xanthomonas spp. are not secreted by the T2S systems from Xcv. Thus, T2S systems from Xanthomonas spp. appear to differ significantly in their substrate specificities.• Transcript analyses revealed that expression of xps genes in Xcv is activated by HrpG and HrpX, key regulators of the T3S system. By contrast, expression of xynC and extracellular protease and xylanase activities are repressed by HrpG and HrpX, suggesting that components and substrates of the Xps system are differentially regulated.

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein–protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition.

Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant-and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.antimicrobials | colicin | EHEC | food safety | plant-made recombinant proteins

Salmonella enterica causes an estimated 1 million illnesses in the United States each year, resulting in 19,000 hospitalizations and 380 deaths, and is one of the four major global causes of diarrhoeal diseases. No effective treatments are available to the food industry. Much attention has been given to colicins, natural non-antibiotic proteins of the bacteriocin class, to control the related pathogen Escherichia coli. We searched Salmonella genomic databases for colicin analogues and cloned and expressed in plants five such proteins, which we call salmocins. Among those, SalE1a and SalE1b were found to possess broad antimicrobial activity against all 99 major Salmonella pathovars. Each of the two salmocins also showed remarkably high potency (>106 AU/µg recombinant protein, or >103 higher than colicins) against major pathogenic target strains. Treatment of poultry meat matrices contaminated with seven key pathogenic serovars confirmed salmocin efficacy as a food safety intervention against Salmonella.

Gram-negative bacteria belonging to the genus Klebsiella are important nosocomial pathogens, readily acquiring resistance to all known antibiotics. Bacteriocins, non-antibiotic antibacterial proteins, have been earlier proposed as potential therapeutic agents for control of other Gram-negative species such as Escherichia, Pseudomonas and Salmonella. This study is the first report describing pore-forming and peptidoglycan-degrading bacteriocins klebicins from Klebsiella. We have identified, cloned, expressed in plants and characterized nine pore-forming and peptidoglycan-degrading bacteriocins from different Klebsiella species. We demonstrate that klebicins can be used for broad and efficient control of 101 of the 107 clinical isolates representing five Klebsiella species, including multi-drug resistant pathovars and pathovars resistant to carbapenem antibiotics.