SummaryGenetic analysis has indicated that the system II pathway for c-type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes ( ccmA-H ) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c 4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli . Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c -haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism ( E. coli ). The ferrochelatase inhibitor N -methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holoCcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo-CcmE as a haem reservoir, a capability system II does not possess.

For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo.With its remarkable ability to adapt to a variety of human tissues, Streptococcus pyogenes (group A streptococcus) provides a unique opportunity to investigate the complex regulatory systems responsible for sensing and responding to environmental changes in the dynamic host environment. Numerous virulence factors have been described that allow this single species of bacterium to produce a wide range of degrees of disease severity and a wide range of clinical manifestations, including pharyngitis and impetigo and invasive diseases such as necrotizing fasciitis, septicemia, and toxic-shock-like syndrome (15). How different streptococcal virulence factors interact with the host to produce these diverse diseases is unknown. However, it is likely that the development of any of these diseases requires that virulence factor expression be highly regulated in an ordered spatial and temporal fashion. Consistent with this, several regulatory factors have been identified which modulate transcription of various virulence genes in response to different environmental cues (reviewed in reference 31). However, the specific signals that are sensed in tissue to control the regulatory network remain largely unknown.Insight into the types of signals that may be sensed in vivo has come mainly from analyses of virulence gene expression by use of in vitro models. Typically, cultures are grown in an artificial medium and the affect of alterations of a specific medium component or growth condition on transcription of genes control...

Regulation of virulence factor expression is critical for pathogenic microorganisms that must sense and adapt to a dynamic host environment; yet, the signal transduction pathways that enable this process are generally poorly understood. Here, we identify LacD.1 as a global regulator of virulence factor expression in the versatile human pathogen, Streptococcus pyogenes. LacD.1 is derived from a class I tagatose‐1,6‐bisphosphate aldolase homologous to those involved in lactose and galactose metabolism in related prokaryotes. However, regulation of transcription by LacD.1 is not dependent on this enzymatic activity or the canonical catabolite repression pathway, but likely does require substrate recognition. Our results suggest that LacD.1 has been adapted as a metabolic sensor, and raise the possibility that regulation of gene expression by metabolic enzymes may be a novel mechanism by which Gram‐positive bacteria, including S. pyogenes, coordinate multiple environmental cues, allowing essential transcription programs to be coupled with perceived nutritional status.

Unlike other cytochromes, c‐type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c‐type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild‐type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N‐terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin‐like protein. Both the B. pertussis dipZ (i.e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol‐reducing compounds, dithiothreitol and 2‐mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435‐amino‐acid periplasmic region.

Isolates of methicillin-resistant Staphylococcus aureus (MRSA) were once linked uniformly with hospital-associated infections; however, community-acquired methicillin-resistant S. aureus (CA-MRSA) now represents an emerging threat worldwide. To examine the association of differential virulence gene expression with outcomes of human infection, we measured transcript abundance of target staphylococcal genes directly in clinical samples from children with active known or suspected CA-MRSA infections. Virulence genes encoding secreted toxins, including Panton-Valentine leukocidin, were highly expressed during superficial and invasive CA-MRSA infections. In contrast, increased expression of surface-associated Protein A was linked only with invasive disease. Comparisons to laboratory growth of corresponding clinical isolates revealed that tissue-specific expression profiles reflect activity of the staphylococcal regulator Agr during human infection. These results represent the first demonstration of staphylococcal gene expression and regulation directly in human tissue. Such analysis will help to unravel the complex interactions between CA-MRSA and its host environmental niches during disease development.