Applied aspects of Rhodococcus genetics

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase

The lpcA locus has been identified in Escherichia coli K12 novobiocin-supersensitive mutants that produce a short lipopolysaccharide (LPS) core which lacks glyceromannoheptose and terminal hexoses. We have characterized lpcA as a single gene mapping around 5.3 min (246 kilobases) on the E. coli K12 chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant plasmids containing only lpcA restored a complete core LPS in the E. coli strain chi711. We show that this strain has an IS5-mediated chromosomal deletion of 35 kilobases that eliminates lpcA. The LpcA protein showed discrete similarities with a family of aldose/ketose isomerases and other proteins of unknown function. The isomerization of sedoheptulose 7-phosphate, into a phosphosugar presumed to be D-glycero-D-mannoheptose 7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA+ and of lpcA mutants containing the recombinant lpcA gene. We concluded that LpcA is the phosphoheptose isomerase used in the first step of glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain glyceromannoheptose in the inner core LPS, indicating that LpcA is an essential component in a conserved biosynthetic pathway of inner core LPS.

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110:identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.

Acetylation of O-specific lipopolysaccharides from Shigella flexneri 3a and 2a occurs in Escherichia coli K-12 carrying cloned S. flexneri 3a and 2a rfb genes

Most of the Shigella flexneri O-specific serotypes result from O-acetyl and/or glucosyl groups added to a common O-repeating unit of the lipopolysaccharide (LPS) molecule. The genes involved in acetylation and/or glucosylation of S. flexneri LPS are physically located on lysogenic bacteriophages, whereas the rfb cluster contains the biosynthesis genes for the common O-repeating unit (D.A.R. Simmons and E. Romanowska, J. Med. Microbiol. 23:289-302, 1987). Using a cosmid cloning strategy, we have cloned the rfb regions from S. flexneri 3a and 2a. Escherichia coli K-12 containing plasmids pYS1-5 (derived from S. flexneri 3a) and pEY5 (derived from S. flexneri 2a) expressed O-specific LPS which reacted immunologically with S. flexneri polyvalent O antiserum. However, O-specific LPS expressed in E. coli K-12 also reacted with group 6 antiserum, indicating the presence of O-acetyl groups attached to one of the rhamnose components of the O-repeating unit. This was confirmed by measuring the amounts of acetate released from purified LPS samples and also by the chemical removal of O-acetyl groups, which abolished group 6 reactivity. The O-acetylation phenotype was absent in an E. coli strain with an sbcB-his-rfb chromosomal deletion and could be restored upon conjugation of F' 129, which carries sequences corresponding to a portion of the deleted region. Our data demonstrate that E. coli K-12 strains possess a novel locus which directs the O acetylation of LPS and is located in the sbcB-rfb region of the chromosomal map.

Relatedness of O-specific lipopolysaccharide side chain genes from strains of Shigella boydii type 12 belonging to two clonal groups and from Escherichia coli O7:K1

The O-specific lipopolysaccharide side chains of Escherichia coli O7 and Shigella boydii type 12 possess similar but not identical chemical structures. We investigated the genetic relatedness between the O-specific side chain genes in members of these two species. Examination of outer membrane protein and lipopolysaccharide (LPS) banding patterns demonstrated that five strains which had been identified as S. boydii type 12 fell into two clonal groups, SB1 and SB2. Hybridizations with O7-specific radiolabeled probes derived from the chromosomal DNA of an E. coli O7 strain detected identical fragments among the three SB1 strains of S. boydii type 12 and the two E. coli O7 reference isolates. The two other S. boydii type 12 strains, which belonged to the SB2 clone, did not show homologies with the O7 probe under high-stringency conditions of hybridization. The homology between the O7 and type 12 LPS gene regions from the SB1 strains was further confirmed by the construction of O-specific side chain-deficient mutations in these strains by homologous recombination of a suicide plasmid containing O7-specific DNA sequences. Immunoblot experiments with O7 antiserum gave a weak cross-reaction with LPS purified from the SB2 strains but a very strong cross-reaction with the LPS from SB1 isolates. Antiserum raised to one of the SB2 strains cross-reacted only with S. boydii type 12 LPS from the SB1 clone but failed to react with O7 LPS.

Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility

We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77. The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different. Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30. No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids. In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids. Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems. The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30.

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.

Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1

The aerobactin-mediated iron uptake system encoded by pColV-K30 and other ColV plasmids has been associated with the ability of Escherichia coli strains to cause disease. We investigated whether the pColV-K30 aerobactin system is present in E. coli K1 VW187 isolated from a human neonate with meningitis. This strain exhibited a functional aerobactin-mediated iron uptake system, as assessed by a cross-feeding bioassay and by its sensitivity to cloacin, a bacteriocin that recognizes the outer membrane receptor for iron-aerobactin complexes. By using a variety of techniques, we could not find any plasmid harboring the aerobactin genes. Hybridization of restriction endonuclease-cleaved chromosomal DNA from strain VW187 with various clones containing subsets of the pColV-K30 aerobactin region showed that the aerobactin genes were located on a 10.5-kilobase-pair chromosomal HindIII restriction fragment which also contained IS1-like insertion sequences. The chromosomal aerobactin region showed a high degree of conservation when compared with the homologous region in plasmid pColV-K30, although it was located on a different restriction endonuclease site environment.

The Escherichia coli transcriptional regulator MarA directly represses transcription of purA and hdeA

The Escherichia coli MarA protein mediates a response to multiple environmental stresses through the activation or repression in vivo of a large number of chromosomal genes. Transcriptional activation for a number of these genes has been shown to occur via direct interaction of MarA with a 20-bp degenerate asymmetric "marbox" sequence. It was not known whether repression by MarA was also direct. We found that purified MarA was sufficient in vitro to repress transcription of both purA and hdeA. Transcription and electrophoretic mobility shift experiments in vitro using mutant promoters suggested that the marbox involved in the repression overlapped the -35 promoter motif and was in the "backward" orientation. This organization contrasts with that of the class II promoters activated by MarA, in which the marbox also overlaps the -35 motif but is in the "forward" orientation. We conclude that MarA, a member of the AraC/XylS family, can act directly as a repressor or an activator, depending on the position and orientation of the marbox within a promoter.

MAD2 downregulation in hypoxia is independent of promoter hypermethylation

Aberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancer cell lines, the mechanism(s) underlying its downregulation remain elusive. The objective of this study was to establish the impact of hypoxia on MAD2 expression and to investigate the potential role of aberrant promoter methylation as a possible mechanism of MAD2 downregulation. For this purpose, three ovarian cancer cell lines, displaying differing levels of MAD2, were treated with chromatin modifying drugs, pre and post-hypoxia exposure and a DHPLC analysis of DNA promoter methylation carried out. We show that hypoxia induces downregulation of MAD2 expression, independently of MAD2 promoter methylation. We also show no evidence of MAD2 promoter methylation in breast and prostate cancer cells or in breast cancer clinical material. While our findings provide no evidence for MAD2 promoter methylation, we show a concomitant upregulation of p21 with downregulation of MAD2 in hypoxia. Our in vitro results were also confirmed in an ovarian cancer tissue microarray (TMA), where a reciprocal staining of MAD2 and CAIX was found in 21/60 (35%) of tumours. In summary, MAD2 downregulation may be a crucial mechanism by which hypoxic cells become chemorefractory. This stems from our previous work where we demonstrated that MAD2 downregulation induces cellular senescence, a viable cellular fate, with resultant cellular resistance to paclitaxel. Moreover, MAD2 downregulation could play a central role in the induction of chemoresistance in hypoxia, a key tumour microenvironment associated with chemoresistance.

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro

BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell’s response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel.
METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches.
RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2k) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8
representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2kcells show enhanced migratory ability. At 72 h after paclitaxel, MAD2kcells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2k MCF7 cell line after paclitaxel reflecting the observed increase in senescence.
CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.

Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (˜2 x 10(-5) of all mRNA). Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.