One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a 'cell wall peptidase' but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

Transfer of the molybdenum cofactor synthesized by Rhodobacter capsulatus MoeA to XdhC and MobA

The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me(2)SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification.     

A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34?kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R.?capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34?kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R.?capsulatus cultures.     

The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)     

Akt/Protein Kinase B Inhibits Cell Death by Preventing the Release of Cytochrome c from Mitochondria

Growth factors signaling through the phosphoinositide 3-kinase/Akt pathway promote cell survival. The mechanism by which the serine/threonine kinase Akt prevents cell death remains unclear. We have previously shown that Akt inhibits the activity of DEVD-targeted caspases without changing the steady-state levels of Bcl-2 and Bcl-x(L). Here we show that Akt inhibits apoptosis and the processing of procaspases to their active forms by delaying mitochondrial changes in a caspase-independent manner. Akt activation is sufficient to inhibit the release of cytochrome c from mitochondria and the alterations in the inner mitochondrial membrane potential. However, Akt cannot inhibit apoptosis induced by microinjection of cytochrome c. We also demonstrated that Akt inhibits apoptosis and cytochrome c release induced by several proapoptotic Bcl-2 family members. Taken together, our results show that Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.     

Molecular Cloning and Expression Analysis of the Rhodobacter capsulatus sodB Gene, Encoding an Iron Superoxide Dismutase

Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.     

蛋白激酶与细胞分化—两种分化诱导剂对酪氨酸蛋白激酶的抑制

在报道视黄酸(RA)是人肝癌细胞株SMMC-7721分化诱导剂的基础上,本文继续报道8-溴-环磷酸腺苷对该细胞也有分化诱导作用,两者都抑制细胞的增殖,降低γ-谷氨酰转肽酶(γ-GT)比活力和升高(ALP)碱性磷酸酶的比活力。在10μmol/LRA和0.5mmol/L-8-Br-cAMP处理细胞1、3、5天后,胞液和膜性组分中的酪氨酸蛋白激酶(TPK)的比活力均降低,其中RA对胞液TPK的作用在早期较明显,约降低30％,而对膜性TPK的影响则随培养天数而逐渐增加,至第5天下降达50％以上。8Br-cAMP则相反,对胞浆TPK的抑制主要发生在3天以后;约抑制43—53％,而对膜性组分则抑制率逐日降低,在第一天较为明显。因TPK是一个细胞增殖恶变的标志,故RA和8-溴-cAMP对TPK的抑制进一步证明这两种分化诱导剂对SMMC-7721细胞的逆转作用。     

Cloning and overexpression of the Rhodobacter capsulatus hemH gene.

In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.     

Effects of Cadmium Stress on Growth,Morphology, and Protein Expression in Rhodobacter capsulatus B10

The effects of cadmium stress on growth, morphology, and protein expression were investigated in Rhodobacter capsulatus B10 using two-dimensional polyacrylamide gel electrophoresis and a scanning electron microscope with an energy dispersive X-ray spectrometer. The bacterium grew in the presence of 150 μM CdCl₂ and highly induced heat-shock proteins (GroEL and Dnak), S-adenosylmethionine synthetase, ribosomal protein S1, aspartate aminotransferase, and phosphoglycerate kinase. Interestingly, the ribosomal protein S1 was proportionally expressed as the amount of cadmium in the medium, suggesting that S1 may be required for the repair of cadmium-mediated cellular damage. On the other hand, we identified five cadmium-binding proteins: 2-methylcitrate dehydratase, phosphate peripalsmic binding protein, inosine-5′-monophosphate dehydrogenase/guanosine-5′-monophosphate reductase, inositol monophosphatase, and lytic murein transglycosylase. The cadmium-treated cells had a filamentous structure and contained less phosphorous than the untreated cells. We propose that these characteristics of the cadmium-treated cells may be due to the inactivation of the phosphate peripalsmic binding protein and lytic murein transglycosylase by cadmium.     

Metabolism of L-Phenylalanine and L-Tyrosine by the Phototrophic Bacterium Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus utilizes the aromatic amino acids L-phenylalanine and L-tyrosine as nitrogen source. L-Phenylalanine is hydroxylated to L-tyrosine, which is further converted into p-hydroxyphenyl pyruvate (pHPP) by a transamination reaction. The bacterium is unable to grow at the expense of these amino acids as the sole carbon source, although it is able to degrade them to homogentisate, probably by unspecific hydroxylation reactions. Metabolization of L-phenylalanine or L-tyrosine as nitrogen source requires phototrophic growth conditions and does not produce free ammonium inside the cells. A low aminotransferase activity with 2-oxoglutarate and L-tyrosine as substrates can be detected in crude extracts of R. capsulatus. Uptake of both amino acids by R. capsulatus was completely inhibited by ammonium addition, which also prevents aminotransferase induction. Received: 21 July 1998 / Accepted: 19 August 1998