The last decade has seen significant advances in our understanding of the physiology, ecology, and molecular biology of chemoautotrophic bacteria. Many ecosystems are dependent on CO2 fixation by either free-living or symbiotic chemoautotrophs. CO2 fixation in the chemoautotroph occurs via the Calvin-Benson-Bassham cycle. The cycle is characterized by three unique enzymatic activities: ribulose bisphosphate carboxylase/oxygenase, phosphoribulokinase, and sedoheptulose bisphosphatase. Ribulose bisphosphate carboxylase/oxygenase is commonly found in the cytoplasm, but a number of bacteria package much of the enzyme into polyhedral organelles, the carboxysomes. The carboxysome genes are located adjacent to cbb genes, which are often, but not always, clustered in large operons. The availability of carbon and reduced substrates control the expression of cbb genes in concert with the LysR-type transcriptional regulator, CbbR. Additional regulatory proteins may also be involved. All of these, as well as related topics, are discussed in detail in this review.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB ؉ plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA ؉ plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA ؉ plasmid but differed over an Ϸ22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA ؉ plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.Rhodococcus equi is a member of the mycolic acid-containing group of actinobacteria, or mycolata, which also includes the Corynebacterium, Gordonia, Mycobacterium, and Nocardia genera (18). Like many other actinomycetes, R. equi is ubiquitous in nature and lives as a saprophyte in soil (25,35,41). The genus Rhodococcus is a genetically diverse taxon that may be empirically classified into two groups (4, 23): the nonpathogenic, or environmental, rhodococci, exemplified by Rhodococcus erythropolis, which includes metabolically versatile bacteria of industrial interest (32), and the pathogenic rhodococci, with two species, the plant pathogen Rhodococcus fascians (22) and the animal pathogen R. equi (25,35,41). All rhodococci typically harbor large conjugative plasmids encoding nicheadaptive functions, such as various primary and secondary metabolic processes in the environmental species and host colonization (virulence) factors in the pathogenic ones. Some of these extrachromosomal replicons are linear megaplasmids of up to 1 Mb in size, whereas others are circular plasmids of Ϸ100 kb (34, 55).R. equi can be isolated from pulmonary and extrapulmonary pyogranulomatous infections in various mammalian hosts. It causes equine purulent bronchopneumonia, or rattles, a severe respiratory disease of foals characterized by extensive abscessation of the lung parenchyma, lymphadenitis, and a high mortality rate. R. equi is also an opportunistic human pathogen associated with AIDS and immunosuppression. Human rho...

Terminal restriction fragment length polymorphism (tRFLP) is a potentially high-throughput method for the analysis of complex microbial communities. Comparison of multiple tRFLP profiles to identify shared and unique components of microbial communities however, is done manually, which is both time consuming and error prone. This paper describes a freely accessible web-based program, T-Align (http://inismor.ucd.ie/~talign/), which addresses this problem. Initially replicate profiles are compared and used to generate a single consensus profile containing only terminal restriction fragments that occur in all replicate profiles. Subsequently consensus profiles representing different communities are compared to produce a list showing whether a terminal restriction fragment (TRF) is present in a particular sample and its relative fluorescence intensity. The use of T-Align thus allows rapid comparison of numerous tRFLP profiles. T-Align is demonstrated by alignment of tRFLP profiles generated from bacterioplankton communities collected from the Irish and Celtic Seas in November 2000. Ubiquitous TRFs and site-specific TRFs were identified using T-Align.

dWe report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species.

Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface IntroductionStreptomycetes are Gram-positive soil bacteria that colonize moist substrates by forming a branched network of multinucleoid hyphae. At some stage during their life cycle, these bacteria are confronted with a hydrophobic environment. For instance, after a feeding substrate mycelium has been established, hyphae leave the aqueous environment to grow into the hydrophobic air. These aerial hyphae differentiate further by forming chains of uninucleoid cells, which metamorphose into pigmented spores. Spores or hyphae of streptomycetes may also encounter hydrophobic solids such as surfaces of dead or living organisms. When streptomycete hyphae leave their aqueous environment, they change their surface. Hyphae in a moist substrate are hydrophilic, whereas the surfaces of aerial hyphae and spores are hydrophobic.Formation of aerial structures has been best studied in Streptomyces coelicolor (for recent reviews, see Chater, 1998;Kelemen and Buttner, 1998;Wösten and Willey, 2000). Bald (bld) mutants of S. coelicolor were isolated that, when grown on rich medium, are affected in the formation of aerial structures and in the production of a small surface-active peptide called SapB (Willey et al., 1991). Many of these mutants appear to be affected in an extracellular signalling cascade involved in the erection of aerial hyphae (Willey et al., 1993;Nodwell et al., 1996;. Experimental evidence suggests the existence of at least five signalling molecules. It was hypothesized that each signal triggers the synthesis and release of the next signal, ultimately leading to the production and secretion of SapB (Willey et al., 1993;Nodwell et al., 1996). By lowering the water surface tension from 72 to 32 mJ m -2 , SapB enables hyphae to breach the water-air interface to grow into the air (Tillotson et al., 1998).Aerial hyphae and spores of S. coelicolor have several surface layers that make them hydrophobic. One surface layer, called the rodlet layer, has a typical ultrastructure of a mosaic of 8-to 10-nm-wide parallel rods (Wildermuth et al., 1971;Smucker and Pfister, 1978). The nature of the surface layers is not known. SapB is not expected to form one of these layers, as this peptide was localized in the culture medium but could not be detected at the surfaces of aerial structures (Wösten and Willey, 2000). SummaryThe filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growi...

The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter flavus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 bp and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. flavus H4-14 showed that transcription of cfxL and cfxS initiated 22 bp upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

The virulence of the intracellular pathogen Rhodococcus equi in foals is dependent on the presence of an 81-kb virulence plasmid encoding the virulence protein VapA. Expression of this protein is induced by exposure to oxidative stress, high temperatures, and low pHs, which reflect the conditions encountered by R. equi when it enters the host environment. The aim of this study was to determine whether the LysR-type transcriptional regulator VirR, which is encoded by the virulence plasmid, is required for the expression of vapA. It was shown that the virR gene is cotranscribed with four downstream genes, one of which encodes a two-component response regulator. The expression of VapA, as monitored by Western blotting, was completely dependent on the presence of virR. Maximal expression was observed when vapA was present together with the complete virR operon, suggesting that at least one of the virR operon genes, in addition to virR, is required for the expression of vapA to wild-type levels. The transcriptional start site of vapA was determined to be a cytidine located 226 bp upstream from the vapA initiation codon. His-tagged VirR protein was expressed in Escherichia coli and purified by nickel affinity chromatography. DNA binding studies showed that purified VirR binds to a DNA fragment containing the vapA promoter. We therefore conclude that VirR is required for the activation of vapA transcription.The gram-positive bacterium Rhodococcus equi is a facultative intracellular pathogen of alveolar macrophages. Although young foals are the primary host of this pathogen, the incidence of R. equi infection in immunocompromised humans has increased markedly over the past 15 years (9,23,46). Infection with R. equi leads to life-threatening pyogranulomatous pneumonia accompanied by gross lesions such as macroabscesses and cavitation (32). The virulence of R. equi in foals is dependent on an indigenous plasmid, which varies in size between 80 and 85 kb (40,42). Plasmid-cured strains are unable to proliferate in macrophages (12,17). A recent analysis of the nucleotide sequences of two virulence plasmids revealed the presence of a 27.5-kb DNA fragment characterized by a significantly lower GϩC content than the remainder of the virulence plasmid (39). The expression of genes located within this region of the virulence plasmid is upregulated following the internalization of R. equi by macrophages, suggesting that this part of the plasmid is a pathogenicity island (33