Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to sef, fim, saf, and tcf, which had been described previously in serotype Typhi, we identified eight new putative chaperoneusher-dependent fimbrial operons, which were termed bcf, sta, stb, ste, std, stc, stg, and sth. Hybridization analysis performed with 16 strains of Salmonella reference collection C and 22 strains of Salmonella reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal Salmonella serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other Salmonella serotypes investigated in that it contained a unique combination of putative fimbrial operons.The genus Salmonella contains pathogens which are closely related genetically but differ in their host range (7). One end of the spectrum is formed by broad-host-range pathogens such as Salmonella enterica serotype Typhimurium, which is frequently associated with cases of disease in a number of animal species, including mice, pigs, poultry, horses, cattle, and sheep (19,43,52). At the other end of the spectrum are pathogens whose ability to cause disease is restricted to a single genus or related genera of vertebrate species. Serotype Typhi is a prototypical host-restricted serotype which causes typhoid fever in humans and higher primates but is unable to produce illness in other vertebrate species. Since there is no inexpensive animal model with which to study serotype Typhi pathogenesis, little is known about virulence factors which are responsible for its apparent adaptation to the human host and its ability to cause typhoid fever. With the sequence of the whole serotype Typhi genome now almost complete, we can begin to address these questions using comparative genomic analysis.One of the virulence factors recently implicated in adaptation of serotype Typhi to the human host is a fimbrial operon termed tcf, for Typhi colonization factor. Serotype Typhi is the only serotype within Salmonella reference collection C (SARC), a strain collection consisting of 16 isolates representing all phylogenetic lineages within the genus Salmonella, which hybridizes with a DNA probe specific to the tcf operon (20). The serotype...

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Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis r , which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.Nisin, a lanthionine-containing peptide produced by certain strains of Lactococcus lactis (24), is widely used in the food industry as a safe and natural preservative (11) because of its antimicrobial activity against a broad range of gram-positive bacteria, including Listeria monocytogenes. Nisin disrupts the cytoplasmic membrane of a bacterial cell through pore formation, which leads to the release of small cytoplasmic compounds, depolarization of the membrane potential, and, ultimately, cell death (4, 5). The lantibiotic uses lipid II as a docking molecule in the target membrane for efficient pore formation. By the binding of nisin to lipid II, bacterial cell wall synthesis is also inhibited (4, 5), which provides another mechanism of cell killing (59).The efficiency of nisin as an antimicrobial agent could be seriously compromised by the occurrence of nisin resistance in spoilage or pathogenic bacteria. The generation of nonstable nisin-resistant (Nis r ) strains of L. lactis under laboratory conditions is achieved relatively easily by stepwise exposure of cells to increasing concentrations of nisin (20,39,42). Since nisin sensitivity and the lipid II content in the cytoplasmic membrane are not directly correlated (29), nisin resistance is expected to be achieved through another mechanism(s).Nisin resistance in bacteria has been associated with an altered fatty acid composition of phospholipids (39,42) or an altered phospholipid composition of the cytoplasmic membrane (41). Lipoteichoic acid (LTA) was shown to play an important role in the nisin re...

Little is known about factors which enable Salmonella serotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present in Salmonella serotypes commonly isolated from livestock and domestic fowl (S. enterica subspecies I) but absent from reptile-associated Salmonella serotypes (S. bongori and S. enterica subspecies II to VII). This DNA region was cloned from Salmonella serotype Typhimurium and sequence analysis revealed the presence of a 6,105-bp open reading frame, designated shdA, whose product's deduced amino acid sequence displayed homology to that of AIDA-I from diarrheagenic Escherichia coli, MisL of serotype Typhimurium, and IcsA of Shigella flexneri. The shdA gene was located adjacent to xseA at 52 min, in a 30-kb DNA region which is not present in Escherichia coli K-12. A serotype Typhimurium shdA mutant was shed with the feces in reduced numbers and for a shorter period of time compared to its isogenic parent. A possible role for the shdA gene during the expansion in host range of S. enterica subspecies I to include warm-blooded vertebrates is discussed.Salmonella serotypes are a frequent constituent of the intestinal flora of poikilothermic animals. The percentage of apparently healthy, cold-blooded vertebrates which harbor Salmonella serotypes ranges from 74 to 94% (20,28,32,34,59), and these bacteria could thus be considered part of the normal intestinal flora (23,48). Salmonella serotypes are also commonly isolated from a fraction (usually Ͻ20%) of warmblooded animal hosts (15,31,49,54). Although chronic carriers, which appear healthy, are observed within the human population and among warm-blooded animals (22,27,35,40), Salmonella serotypes are commonly associated with illness in these hosts (55). Consequently Salmonella serotypes are regarded as pathogens rather than part of the normal intestinal flora of homeothermic animals.On the basis of multilocus enzyme electrophoresis and comparative sequence analysis of orthologous genes, two species, S. enterica and S. bongori, have been assigned to the genus Salmonella (18,46). S. enterica is further subdivided into seven subspecies designated with roman numerals (18, 44). While S. bongori and S. enterica subspecies II, IIIa, IIIb, IV, VI, and VII are mainly associated with cold-blooded vertebrates, members of S. enterica subspecies I are frequently isolated from avian and mammalian hosts (44). For instance, of the 90,201 Salmonella isolates collected between 1977 and 1992 by the German National Reference Center for Enteric Pathogens from humans and warm-blooded animals, 89,798 isolates (99.55%) belonged to S. enterica subspecies I (1). Currently it is not clear which virulence mechanisms are responsible for the apparent adaptation of S. enterica subspecies I to circulation within populations of warm-blooded animals.S. bongori or S. enterica subspecies II to VII are able to infect humans, colonize the intestine and cause disease (1). Human infections with serotypes of S. bongori and S. enterica su...

BackgroundIn research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed.ResultsA number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance.ConclusionEven in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.

Lipid II is essential for nisin-mediated pore formation at nano-molar concentrations. We tested whether nisin resistance could result from different Lipid II levels, by comparing the maximal Lipid II pool in Micrococcus flavus (sensitive) and Listeria monocytogenes (relatively insensitive) and their nisin-resistant variants, with a newly developed method. No correlation was observed between the maximal Lipid II pool and nisin sensitivity, as was further corroborated by using spheroplasts of nisin-resistant and wild-type strains of M. flavus, which were equally sensitive to nisin.

This is the first time that a genomic transcriptional lacZ reporter gene H. pylori library has been used as a tool for the fast and efficient identification of environmental stress-regulated H. pylori genes.