The "Bacterial Reverse Mutation Assay" is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX (R)(1) system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537.TA1538.TA102, WP2uvrApKM101) and descendants of TA98 (YG1021,YG1024,YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98,TA100 and their YG descendant strains as well as by WP2uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette. (C) 2008 Elsevier B.V. All rights reserved.

The evaluation of the mutagenic activity of cigarette smoke is mostly based on studies with condensates or extracts in the standard Ames assay. These samples only insufficiently reflect the composition of the actual generated aerosol. Therefore, such atmospheres should be analysed in their native composition to gain a real signal of its mutagenic capacity. Based on the technical difficulties of testing native air contaminants, there are no accepted methods for effective exposure of bacteria under such conditions. Therefore, we established a new experimental approach for direct exposure of bacteria in a modified CULTEX® system (Patent no. DE 19801763/PCT/EP99/00295) connected to a smoking machine. This allowed us to investigate the mutagenic activity of native mainstream smoke of the research cigarette K2R4F by exposure of Salmonella Typhimurium strains. In comparison to studies using the plate incorporation assay, the direct exposure of bacteria to smoke on the agar surface enhances contact to the aerosols. By using this modification of the Ames assay, we demonstrate that it is possible to analyse the effects of native whole smoke and the gas vapour phase of cigarettes directly and achieve a dose-dependent induction of revertants. In a number of experiments, the treatment of strains TA98 and TA100 with whole smoke and the gas vapour phase of K2R4F cigarettes resulted in the induction of revertants dependent on the dilution of smoke and the number of cigarettes smoked. Our alternative procedure of exposing bacteria directly to gases and complex mixtures offers new possibilities in the field of inhalation genotoxicology for the evaluation of genotoxicity in the Ames assay.

Helicobacter pylori infection of humans is so old that its population genetic structure reflects that of ancient human migrations. A closely related species, Helicobacter acinonychis, is specific for large felines, including cheetahs, lions, and tigers, whereas hosts more closely related to humans harbor more distantly related Helicobacter species. This observation suggests a jump between host species. But who ate whom and when did it happen? In order to resolve this question, we determined the genomic sequence of H. acinonychis strain Sheeba and compared it to genomes from H. pylori. The conserved core genes between the genomes are so similar that the host jump probably occurred within the last 200,000 (range 50,000 - 400,000) years. However, the Sheeba genome also possesses unique features that indicate the direction of the host jump, namely from early humans to cats. Sheeba possesses an unusually large number of highly fragmented genes, many encoding outer membrane proteins, which may have been destroyed in order to bypass deleterious responses from the feline host immune system. In addition, the few Sheeba-specific genes that were found include a cluster of genes encoding sialylation of the bacterial cell surface carbohydrates, which were imported by horizontal genetic exchange and might also help to evade host immune defenses. These results provide a genomic basis for elucidating molecular events that allow bacteria to adapt to novel animal hosts.

Helicobacter pylori, one of the most common bacterial pathogens, colonizes the human stomach and causes a variety of gastric diseases. This pathogen elicits a range of phenotypic responses in infected cultured AGS gastric epithelial cells, including expression of proinflammatory genes and changes in the actin cytoskeleton. Some of these responses are mediated by the type IV secretion system (T4SS) encoded by the cag pathogenicity island. We have used two global approaches, namely 2-DE combined with PMF and cDNA expression array analyses, to study in both a comprehensive and quantitative manner the protein profile and the temporal patterns of mRNA accumulation in AGS cells upon infection with H. pylori and isogenic T4SS mutants. We identified 140 transcripts and detected 190 protein species that were differentially regulated upon infection. Infection with wild-type H. pylori induced expression of a variety of host genes and changes in protein pattern involved in transcriptional responses, cell shape regulation and signal transduction. Among them, some were differentially regulated in a cag PAI-dependent manner, as shown by both the proteomic and cDNA expression array approaches. While 2-DE and PMF allowed us to examine the protein profiles in the infected host, array analysis enabled us to demonstrate dynamic temporal changes in host gene expression profile. In conclusion, our combined application of the two global approaches provides further molecular details on how the host cell responds to infection by H. pylori and its isogenic T4SS mutants on both transcriptional and protein levels. The findings pinpoint host proteins such as serine/threonine and tyrosine kinases, transcription factors, cell cycle related components and actin cytoskeletal signaling molecules as potential targets of individual H. pylori virulence determinants. This study serves as a basis for future work on transcription and proteome analyses of the H. pylori infection model.

Sequence diversity and gene content distinguish most isolates of Helicobacter pylori. Even greater sequence differences differentiate distinct populations of H. pylori from different continents, but it was not clear whether these populations also differ in gene content. To address this question, we tested 56 globally representative strains of H. pylori and four strains of Helicobacter acinonychis with whole genome microarrays. Of the weighted average of 1,531 genes present in the two sequenced genomes, 25% are absent in at least one strain of H. pylori and 21% were absent or variable in H. acinonychis. We extrapolate that the core genome present in all isolates of H. pylori contains 1,111 genes. Variable genes tend to be small and possess unusual GC content; many of them have probably been imported by horizontal gene transfer. Phylogenetic trees based on the microarray data differ from those based on sequences of seven genes from the core genome. These discrepancies are due to homoplasies resulting from independent gene loss by deletion or recombination in multiple strains, which distort phylogenetic patterns. The patterns of these discrepancies versus population structure allow a reconstruction of the timing of the acquisition of variable genes within this species. Variable genes that are located within the cag pathogenicity island were apparently first acquired en bloc after speciation. In contrast, most other variable genes are of unknown function or encode restriction/modification enzymes, transposases, or outer membrane proteins. These seem to have been acquired prior to speciation of H. pylori and were subsequently lost by convergent evolution within individual strains. Thus, the use of microarrays can reveal patterns of gene gain or loss when examined within a phylogenetic context that is based on sequences of core genes.

The flagellar system of Helicobacter pylori, which comprises more than 40 mostly unclustered genes, is essential for colonization of the human stomach mucosa. In order to elucidate the complex transcriptional circuitry of flagellar biosynthesis in H. pylori and its link to other cell functions, mutants in regulatory genes governing flagellar biosynthesis (rpoN, flgR, flhA, flhF, HP0244) and whole-genome microarray technology were used in this study. The regulon controlled by RpoN, its activator FlgR (FleR) and the cognate histidine kinase HP0244 (FleS) was characterized on a genome-wide scale for the first time. Seven novel genes (HP1076, HP1233, HP1154/1155, HP0366/367, HP0869) were identified as belonging to RpoN-associated flagellar regulons. The hydrogenase accessory gene HP0869 was the only annotated non-flagellar gene in the RpoN regulon. Flagellar basal body components FlhA and FlhF were characterized as functional equivalents to master regulators in H. pylori, as their absence led to a general reduction of transcripts in the RpoN (class 2) and FliA (class 3) regulons, and of 24 genes newly attributed to intermediate regulons, under the control of two or more promoters. FlhA- and FlhF-dependent regulons comprised flagellar and non-flagellar genes. Transcriptome analysis revealed that negative feedback regulation of the FliA regulon was dependent on the antisigma factor FlgM. FlgM was also involved in FlhA- but not FlhF-dependent feedback control of the RpoN regulon. In contrast to other bacteria, chemotaxis and flagellar motor genes were not controlled by FliA or RpoN. A true master regulator of flagellar biosynthesis is absent in H. pylori, consistent with the essential role of flagellar motility and chemotaxis for this organism.