A regulatory network of Sinorhizobium meliloti genes involved in adaptation to iron-limiting conditions and the involvement of the rhizobial iron regulator gene (rirA) were analyzed by mutation and microarray analyses. A constructed S. meliloti rirA mutant exhibited growth defects and enhanced H 2 O 2 sensitivity in the presence of iron, but symbiotic nitrogen fixation was not affected. To identify iron-responsive and RirA-regulated S. meliloti genes, a transcriptome approach using whole-genome microarrays was used. Altogether, 45 genes were found to be jointly derepressed by mutation of rirA and under different iron-limited conditions. As expected, a number of genes involved in iron transport (e.g., hmuPSTU, shmR, rhbABCDEF, rhtX, and rhtA) and also genes with predicted functions in energy metabolism (e.g., fixN3, fixP3, and qxtAB) and exopolysaccharide production (e.g., exoY and exoN) were found in this group of genes. In addition, the iron deficiency response of S. meliloti also involved rirA-independent expression changes, including repression of the S. meliloti flagellar regulon. Finally, the RirA modulon also includes genes that are not iron responsive, including a gene cluster putatively involved in Fe-S cluster formation (sufA, sufS, sufD, sufC, and sufB).

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The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoteroperator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

The transcriptional regulators RamA, RamB and GlxR were detected to bind to the promoter region of the resuscitation promoting factor 2 (rpf2) gene involved in growth and culturability of Corynebacterium glutamicum. DNA-binding sites were identified by bioinformatic analysis and verified by electrophoretic mobility shift assays with purified hexahistidyl-tagged proteins. Carbon source-dependent deregulation of rpf2 expression was demonstrated in vivo in ramA and ramB mutants and in a C. glutamicum strain overexpressing glxR. The deduced network of regulatory interactions provided insights into the complex regulation pattern of rpf2 expression in C. glutamicum.

bThe invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system (T3SS) and the SPI4-encoded adhesin SiiE. The invasion of polarized cells is more efficient than that of nonpolarized cells, and we observed the formation of clusters of bacteria on infected cells. Here we demonstrate that the invasion of polarized cells is a highly cooperative activity. Using a novel live-cell imaging approach, we visualized the cooperative entry of multiple bacteria into ruffles induced on the apical surfaces of polarized cells. The induction of membrane ruffles by activity of Salmonella enables otherwise noninvasive mutant strains to enter polarized host cells. Bacterial motility and chemotaxis were of lower importance for cooperativity in polarized-cell invasion. We propose that cooperative invasion is a key factor for the very efficient entry into polarized cells and a factor contributing to epithelial damage and intestinal inflammation.

Secreted proteins of the human pathogen Corynebacterium diphtheriae might be involved in important pathogen-host cell interactions. Here, we present the first systematic reference map of the extracellular and cell surface proteome fractions of the type strain C. diphtheriae C7s(-)tox-. The analysis window of 2-DE covered the pI range from 3 to 10 along with a MW range from 8 to 150 kDa. Computational analysis of the 2-D gels detected almost 150 protein spots in the extracellular proteome fraction and about 80 protein spots of the cell surface proteome. MALDI-TOF-MS and PMF with trypsin unambiguously identified 107 extracellular protein spots and 53 protein spots of the cell surface, representing in total 85 different proteins of C. diphtheriae C7s(-)tox-. Several of the identified proteins are encoded by pathogenicity islands and might represent virulence factors of C. diphtheriae. Additionally, four solute-binding proteins (HmuT, Irp6A, CiuA, and FrgD) of different iron ABC transporters were identified, with the hitherto uncharacterized FrgD protein being the most abundant one of the cell surface proteome of C. diphtheriae C7s(-)tox-.

Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella -induced filaments (SIFs) connected to the Salmonella -containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as Δ ssaV . A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), Δ sseF or Δ ssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM Δ sseF and Δ ssaV compared to WT. In addition, STM Δ ssaV , but not Δ sseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms.

Adhesins are crucial virulence factors of pathogenic bacteria involved in colonization, transmission and pathogenesis. Many bacterial genomes contain the information for a surprisingly large number of diverse adhesive structures. One prominent example is the invasive and facultative intracellular pathogen Salmonella enterica with an adhesiome of up to 20 adhesins. Such large repertoire of adhesins contributes to colonization of a broad range of host species and may allow adaptation to various environments within the host, as well as in non-host environments. For S. enterica, only few members of the adhesiome are functionally expressed under laboratory conditions, and accordingly the structural and functional understanding of the majority of adhesins is sparse. We have devised a simple and versatile approach to functionally express all adhesins of S. enterica serotype Typhimurium, either within Salmonella or within heterologous hosts such as Escherichia coli. We demonstrate the surface expression of various so far cryptic adhesins and show ultrastructural features using atomic force microscopy and transmission electron microscopy. In summary, we report for the first time the expression of the entire adhesiome of S. enterica serotype Typhimurium.