Manisha Dosanjh

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1s 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O2-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

Catabolism of Benzoate and Phthalate in Rhodococcus sp. Strain RHA1: Redundancies and Convergence

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the beta-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the beta-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent beta-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

WhiB7, an Fe-S-dependent Transcription Factor That Activates Species-specific Repertoires of Drug Resistance Determinants in Actinobacteria

Background: WhiB7 is essential for antibiotic resistance in M. tuberculosis. Results: WhiB7 requires conserved residues, including a redox-sensitive center and DNA-binding motif, to coordinate transcription of species-specific drug resistance genes in diverse Actinobacteria. Conclusion: WhiB7 activates species-specific drug resistance genes in Actinobacteria. Significance: Understanding WhiB7 activity may allow the development of drugs that sensitize bacteria to antibiotics. WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the hosts repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimers absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.

Proteomic Analysis of Survival of Rhodococcus jostii RHA1 during Carbon Starvation

ABSTRACT Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ∼250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO2 utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.

Hyaluronan binding identifies the most proliferative activated and memory T cells

CD44 is expressed on T cells where its ability to bind hyaluronan is tightly regulated. Here, we investigated when T cells bind hyaluronan during an immune response. We found that naïve, murine T cells do not bind fluoresceinated hyaluronan but are induced to bind upon antigen‐induced T‐cell activation in vitro and in vivo. Hyaluronan binding occurred on proliferating T cells and the percentage of hyaluronan‐binding cells correlated with the strength of the activation stimulus. A small percentage of hyaluronan‐binding cells persisted after in vitro activation and had a memory phenotype (CD122+CD44hi). This hyaluronan‐binding population increased after culture with IL‐7 or IL‐15 and proliferated more rapidly than nonbinding cells. In vivo, approximately 20–30% of antigen‐specific OT‐I CD8+ memory T cells in the spleen and BM bound hyaluronan. Hyaluronan binding identified memory cells that proliferated faster in IL‐7 and IL‐15, and enriched for CD62L+ central memory cells. In vivo homeostatic proliferation induced hyaluronan binding on a small percentage of the most rapidly dividing cells after several cell divisions. This study demonstrates that hyaluronan binding is induced upon antigen‐induced T‐cell activation and occurs on a percentage of the most proliferative activated and memory T cells.

Endotoxin free hyaluronan and hyaluronan fragments do not stimulate TNF-α, interleukin-12 or upregulate co-stimulatory molecules in dendritic cells or macrophages

The extracellular matrix glycosaminoglycan, hyaluronan, has been described as a regulator of tissue inflammation, with hyaluronan fragments reported to stimulate innate immune cells. High molecular mass hyaluronan is normally present in tissues, but upon inflammation lower molecular mass fragments are generated. It is unclear if these hyaluronan fragments induce an inflammatory response or are a consequence of inflammation. In this study, mouse bone marrow derived macrophages and dendritic cells (DCs) were stimulated with various sizes of hyaluronan from different sources, fragmented hyaluronan, hyaluronidases and heavy chain modified-hyaluronan (HA-HC). Key pro-inflammatory molecules, tumour necrosis factor alpha, interleukin-1 beta, interleukin-12, CCL3, and the co-stimulatory molecules, CD40 and CD86 were measured. Only human umbilical cord hyaluronan, bovine testes and Streptomyces hyaluronlyticus hyaluronidase stimulated macrophages and DCs, however, these reagents were found to be contaminated with endotoxin, which was not fully removed by polymyxin B treatment. In contrast, pharmaceutical grade hyaluronan and hyaluronan fragments failed to stimulate in vitro-derived or ex vivo macrophages and DCs, and did not induce leukocyte recruitment after intratracheal instillation into mouse lungs. Hence, endotoxin-free pharmaceutical grade hyaluronan does not stimulate macrophages and DCs in our inflammatory models. These results emphasize the importance of ensuring hyaluronan preparations are endotoxin free.

Characterization of a mycothiol ligase mutant of Rhodococcus jostii RHA1

Mycothiol (1d-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-alpha-D-glucopyranoside) is an important microbial thiol present only in actinomycetes. Rhodococcus jostii RHA1 degrades a wide range of xenobiotics, including polychlorinated biphenyls, nitriles and N-nitrosodimethylamine. Analyses revealed that this strain produces two thiols, mycothiol and ergothioneine, found in the other actinomycetes. A mycothiol ligase mutant strain of R. jostii RHA1 deficient in the production of mycothiol was constructed. This mutant has a number of interesting characteristics: (a) it overproduces the intermediate glucosamine-inositol (1-O-(2-amino-1-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol); (b) it is deficient in the biochemical degradation of a number of xenobiotics metabolized by the parent strain; (c) it shows increased susceptibility to a number of antibiotics; and (d) it shows unusual growth characteristics, exhibiting a long lag phase before normal exponential growth. The diverse phenotypes of the mutant indicate the utility of R. jostii RHA1 as a model for deciphering the various functions of mycothiol.

Hyaluronan Binding Identifies a Functionally Distinct Alveolar Macrophage–like Population in Bone Marrow–Derived Dendritic Cell Cultures

Although classical dendritic cells (DCs) arise from distinct progenitors in the bone marrow, the origin of inflammatory DCs and the distinction between monocyte-derived DCs and macrophages is less clear. In vitro culture of mouse bone marrow cells with GM-CSF is a well-established method to generate DCs, but GM-CSF has also been used to generate bone marrow–derived macrophages. In this article, we identify a distinct subpopulation of cells within the GM-CSF bone marrow–derived DC culture based on their ability to bind hyaluronan (HA), a major component of the extracellular matrix and ligand for CD44. HA identified a morphologically distinct subpopulation of cells within the immature DC population (CD11c+ MHC IImid/low) that were CCR5+/CCR7− and proliferated in response to GM-CSF, but, unlike immature DCs, did not develop into mature DCs expressing CCR7 and high levels of MHC II, even after stimulation with LPS. The majority of these cells produced TNF-α in response to LPS but were unable to activate naive T cells, whereas the majority of mature DCs produced IL-12 and activated naive T cells. This HA binding population shared many characteristics with alveolar macrophages and was retained in the alveolar space after lung instillation even after LPS stimulation, whereas the MHC IIhigh mature DCs were found in the draining lymph node. Thus, HA binding in combination with MHC II expression can be used to identify alveolar-like macrophages from GM-CSF–treated bone marrow cultures, which provides a useful in vitro model to study alveolar macrophages.

The survival of fetal and bone marrow monocyte-derived alveolar macrophages is promoted by CD44 and its interaction with hyaluronan

INTRODUCTION The airways are primary entry sites for pathogens and airborne particles where alveolar macrophages (AM^) are a first line of defense to phagocytose pathogens, particulates, and cell debris. Shortly after birth, colony stimulating factor-2 (CSF2 or granulocyte-macrophage-CSF) drives the differentiation of fetal monocytes into long-lived AM^ capable of selfrenewal. Their self-renewal is sufficient to maintain the AM^ population at homeostasis and does not require replenishment from bone marrow (BM)-derived monocytes. However, BM-derived cells are capable of developing into AM^, as after AM^ ablation by irradiation, subsequent BM reconstitution leads to BM-derived AM^ in adult mice. Yolk sac macrophages, fetal liver, and adult monocytes can colonize the lungs of Csf2rb / mice and develop into functional AM^, showing that progenitors of different origins can differentiate into AM^, raising the possibility that it is the alveolar environment, not the origin of the progenitor, that determines their differentiation into AM^. In acute inflammation, the numbers of AM^ are reduced and then restored after inflammation is resolved. In influenza infection, the recovery of AM^ was attributed to AM^ self-renewal, whereas 2 months after acute lipopolysaccharide (LPS)induced inflammation, the majority of AM^ were shown to be BM derived. Thus, in adult mice, the extent to which monocytes contribute to AM^ renewal after inflammation is not clear, nor are the factors that regulate monocyte differentiation and AM^ renewal after inflammation. Such information would considerably aid the understanding of the AM^ renewal process that could be crucial for the resolution of inflammation and return to homeostasis. At homeostasis, AM^ are thought to exist in a somewhat immunosuppressive environment where they phagocytose pathogens without alerting other innate immune cells. If they cannot clear the infection, they initiate innate immune responses: for example, in response to Streptococcus

CD45 regulates GM-CSF, retinoic acid and T-cell homing in intestinal inflammation

CD45 is a leukocyte-specific tyrosine phosphatase important for T-cell development, and as a result, CD45−/− mice have substantially reduced numbers of T cells. Here we show that, upon dextran sodium sulfate (DSS)-induced colitis, CD45−/− mice have equivalent intestinal pathology and T-cell numbers in their colon as C57BL/6 mice and show enhanced weight loss. CD45−/− mice have a greater percentage of α4β7+ T cells prior to and after colitis and an increased percentage of T cells producing inflammatory cytokines in the inflamed colon, suggesting that CD45−/− effector T cells preferentially home to the intestine. In DSS-induced colitis in CD45RAG−/− mice lacking an adaptive immune system, CD45 was required for optimal granulocyte-macrophage colony-stimulating factor (GM-CSF) and retinoic acid (RA) production by innate immune cells. Addition of CD45+/+ T cells led to greater weight loss in the RAG−/− mice compared with CD45RAG−/− mice that correlated with reduced α4β7+ T cells and lower recruitment to the colon of CD45RAG−/− mice in DSS-induced colitis. Addition of exogenous GM-CSF to CD45RAG−/− mice rescued RA production, increased colonic T-cell numbers, and increased weight loss. This demonstrates opposing effects of CD45 in innate and adaptive immune cells in proinflammatory responses and the expression of the gut-homing molecule, α4β7.