Figure 3 of this Letter contains an inadvertently duplicated panel: the PBS 30 panel is identical to the aGalCer panel (top right). The corrected panels are shown here. Our results and conclusions are unaffected by this oversight. CORRIGENDUM

Mycobacterium tuberculosis secretes ESAT-6, a virulence factor that triggers cell-mediated immune responses and IFN-␥ production during tuberculosis. ESAT-6 is transported across the bacterial envelope by a specialized secretion system with a FSD (FtsK-SpoIIIE domain) membrane protein. Although the presence of ESAT-6-like genes has been identified in the genomes of other microbes, the possibility that they may encode general virulence functions has hitherto not been addressed. Herein we show that the human pathogen Staphylococcus aureus secretes EsxA and EsxB, ESAT-6-like proteins, across the bacterial envelope. Staphylococcal esxA and esxB are clustered with six other genes and some of these are required for synthesis or secretion of EsxA and EsxB. Mutants that failed to secrete EsxA and EsxB displayed defects in the pathogenesis of S. aureus murine abscesses, suggesting that this specialized secretion system may be a general strategy of human bacterial pathogenesis.specialized secretion ͉ Gram-positive ͉ exoprotein ͉ ess

Although bacteria use different iron compounds in vitGro, the possibility that microbes distinguish between these iron sources during infection has hitherto not been examined. We applied stable isotope labeling to detect source-specific iron by mass spectrometry and show that Staphylococcus aureus preferentially imports heme iron over transferrin iron. By combining this approach with computational genome analysis, we identified hts (heme transport system), a gene cluster that promotes preferred heme iron import by S. aureus. Heme iron scavenging by means of hts is required for staphylococcal pathogenesis in animal hosts, indicating that heme iron is the preferred iron source during the initiation of infection.

The plague is caused by the bacterium Yersinia pestis. Plague bacteria are thought to inject effector Yop proteins into host cells via the type III pathway. The identity of the host cells targeted for injection during plague infection is unknown. We found, using Yop β-lactamase hybrids and fluorescent staining of live cells from plague-infected animals, that Y. pestis selected immune cells for injection. In vivo, dendritic cells, macrophages, and neutrophils were injected most frequently, whereas B and T lymphocytes were rarely selected. Thus, it appears that Y. pestis disables these cell populations to annihilate host immune responses during plague.Yersinia pestis, the causative agent of plague or black death, harbors a virulence plasmid that encodes a type III secretion machine and its Yop protein substrates (1, 2). The essential contribution of type III secretion to the pathogenesis of plague was revealed by comparing lethal infectious doses of wild-type and mutant strains (3). Yersinia type III injection of Yop proteins into tissue culture cells has been detected with fluorescent microscopy, adenylate cyclase or Elk tag fusions, and fractionation techniques (4-7). These technologies, however, have not been useful for measuring the selection of host cells as targets of type III injection during infection.CCF2-AM, a β-lactamase substrate, has been used to detect bacterial type HI reporter injection into tissue culture cells(8). CCF2-AM is a membrane-permeant ester with two fluorophores attached to cephalosporin that exhibit fluorescence resonance energy transfer (FRET). Excitation of coumarin (409 nm) results in green fluorescence emission from fluorescein (520 nm) in intact CCF2-AM (9). β-Lactamase cleaves CCF2-AM, thereby disrupting FRET and establishing blue fluorescence emission. To investigate the usefulness of β-lactamase as a reporter for in vivo target cell selection by Y. pestis, we transformed plasmids carrying translational fusions between YopM or glutathione S-transferase (GST) and the mature domain of TEM-1 β-lactamase (YopM-Bla and GST-Bla, respectively) into Y. pestis strain KIM D27(10) Bacterial cultures were induced for type III secretion via the depletion of calcium at 37°C and then centrifuged to separate the extracellular medium from bacterial cells (11). Immunoblotting of cell-associated and secreted proteins identified YopM-Bla in the extracellular medium of KIM D27 cultures (Fig. 1A). In contrast, GST-Bla or RpoA, the α subunit of RNA polymerase, were not secreted. Disruption of yscU, which encodes a secretion machine component, abrogates all type III secretion (12).

Microbial populations in complex environmental samples are difficult to characterize; current techniques are incomplete and time consuming. We investigated a polymerase chain reaction (PCR)-based method for rapidly comparing bacterial communities independent of culture or cloning. Community 16S rRNA genes were amplified and fluorescently labeled by PCR. The labeled products were digested by a restriction enzyme and the labeled, terminal restriction fragments (TRFs) were separated by electrophoresis and detected by laser-induced fluorescence on an automated gene sequencer. PCR parameters were optimized using an in vitro model community of known organisms. Community comparisons were made between deer fecal pellets, petroleum hydrocarbon-contaminated sands and pristine sand. Principal components analysis (PCA) was used to compare the resulting TRF patterns (TRFPs). Patterns derived from a single enzyme digest did not result in accurate community characterizations. Accurate characterizations reflecting the expected bacterial community biology were only achieved by combining TRFP data derived from different enzyme digestions. Suggestions are offered for future use of this technique.