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The effects of copper (Cu) on trace metal and antibiotic resistance of Pseudomonas aeruginosa have been investigated. Cu treatments induced resistance not only to this metal but also, surprisingly, to zinc (Zn). Quantitative reverse transcription-PCR (qRT-PCR) revealed that after Cu treatment the transcription of the czcRS two-component system (TCS) operon was enhanced as well as that of the czcCBA operon encoding an efflux pump specific for zinc, cadmium, and cobalt. Cu treatments at the same time caused a decrease in the production of OprD porin, resulting in resistance to the carbapenem antibiotic imipenem. The CzcR regulator was known to repress oprD. However, Cu was still able to decrease the production of OprD and induce imipenem resistance in a czcRS knockout mutant. This strongly suggested that another Cu-dependent regulatory system was acting negatively on oprD expression. TCS regulator genes copR-copS have been shown to be involved in Cu tolerance in P. aeruginosa, qRT-PCR showed that overproduction of the CopR or of the CzcR regulator resulted in increased transcription of the czcC gene as well as in a decrease in oprD gene transcription, either in the wild-type strain or in the czcRS knockout mutant. Overproduction experiments suggest that a metal-dependent mechanism operates at the posttranscriptional level to control the production of the CzcCBA efflux pump. This study shows that CopR is a new negative regulator of OprD porin and that it links Zn, Cu, and imipenem resistances by interacting with the CzcRS TCS.

Bacillus subtilis has multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containing ssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for the secG-yvaK-rnr-smpB-ssrA cluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR and lacZ fusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One [[sigma].sup.A]-like promoter preceded secG ([P.sub.1]), and internal [[sigma].sup.A]-like promoters were found in both the rnr.smpB ([P.sub.2]) and smpB-ssrA intervals ([P.sub.3] and [P.sub.HS]). Another internal promoter lay in the secG-yvaK intercistronic region, and this activity ([P.sub.B]) was dependent on the general stress factor [[sigma].sup.B]. Null mutations in the four genes downstream from [P.sub.B] were tested for their effects on growth. Loss of yvaK (carboxylesterase E) or rnr (RNase R) caused no obvious phenotype. By contrast, smpB was required for growth at high temperature (52[degrees]C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably, smpB and ssrA were also required for growth at low temperature (16[degrees]C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role of ssrA and indicate that the ribosome rescue system is important at both extremes of the B. subtilis temperature range. doi:10.1128/JB.00062-.07

Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 [+ or -] 10.8 nm long) are usually in a lateral position on the cells. Fibriilar ([Fib.sup.+]) and nonfibrillar ([Fib.sup.-]) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% [Fib.sup.-] cells and the less hydrophobic cells from the buffer phase entirely comprised [Fib.sup.-] cells. The [Fib.sup.+] and [Fib.sup.-] subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, [Fib.sup.+], and [Fib.sup.-] cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and [Fib.sup.+] cells that were absent from the [Fib.sup.-] cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogoid staining with anti-Aap antibodies labeled tuft fibrils of the WT and [Fib.sup.+] cells but not the cell surface of Fibcells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and [Fib.sup.+] S. epidermidis NCTC 11047 populations. Reverse transcriptionPCR showed that aap was transcribed in both [Fib.sup.+] and [Fib.sup.-] cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

Drug exporters contribute to the intrinsic drug resistance in many organisms. Although there are at least 20 exporter genes in Escherichia coli, most of them apparently do not confer drug resistance in complex laboratory media except for the AcrAB, EmrE, and MdfA efflux systems. In this study, we comprehensively investigated the growth phase-dependent expression of drug exporter genes. The expression of acrAB, emrAB, emrD, emrE, emrKY, mdfA, and ydgFE is stable at moderate levels during any growth phase, whereas mdtEF promoter activity greatly increased with cell growth and reached the maximum level at the late stationary phase. The growth phase-dependent increase in mdtEF expression was also observed on quantitative reverse transcription-PCR analysis. As expected from the transporter expression, the stationary-phase cells actually showed MdtEF-dependent tolerance to drugs and toxic dyes. Growth phase-dependent elevation of mdtEF expression was found to be mediated by the stationary-phase [sigma] factor rpoS and the RpoS-dependent signaling pathway, Hfq, GadY, and GadX. The induction level was decreased by tnaAB deletion, suggesting that indole sensing stimulates this process.

The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems, mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.

The P2-1ike coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.

The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26[degrees]C lacked the 45-kDa flageilin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) orfliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.

In the murine model of infection, a Mycobacterium tuberculosis mce1 operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the mce1 genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the mce1 genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of mce1 expression, we created an M. tuberculosis mce1R deletion mutant. There was no difference in expression of mce1 operon genes in [DELTA]mce1R compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of mce1 genes was significantly higher in [DELTA]mce1R. In addition, overexpression of mce1R resulted in repression of the mce1 genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the mce1 operon. We propose that Mce1R facilitates balanced temporal expression of the mce1 products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of M. tuberculosis.

The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extra-cellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.

Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. The aims of the investigation were to develop and test a sensitive and reproducible method for the study of gene expression in Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls.

The study developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII noroviruses (NoV) in fecal samples, as well as shellfish samples. These assays provide rapid, sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical ad shellfish samples.

Research was conducted to illustrate the synthesis of multiple extracellular lipases in Candida rugosa. To quantify the expression of lip genes, a competitive reverse transcription-PCR assay was developed. Using the approach made it possible to demonstrate the differential expression of the five lip genes in the presence of different inducers that are known to be able to increase C rugosa lipase production.

A reverse transcription-PCR assay was developed to investigate the expression of Tri5 by Fusarium species in planta and in vitro. An increase in Tri5 expression was observed following treatment of Fusarium culmorum with fungicides. An inverse relationship between Tri5 expression and biomass was also observed. Results provide an enhanced understanding of the influence of different host tissues and chemical control agents on trichothecene production.

The reverse transcriptase/polymerase chain reaction (RT-PCR) process is examined with particular attention to the common observation that control DNA reaction mixtures with RT do not provide DNA amplification during PCR while control DNA reaction mixtures without RT do not inhibit PCR. The findings indicate that the RT interaction with the specific mRNA or cDNA template mediates RT inhibition of the PCR and that such inhibitory effect is influenced by template concentration.

A triplex reverse transcriptase PCR (RT-PCR), developed to detect hepatitis A virus and rotavirus is cost effective and saves time. The triplex PCR requires primer concentrations to be optimized for efficient detection of organisms in a mixed sample. This technology finds application in clinical diagnosis and study of environmental samples.

The applicability of selected enteric viral surrogates in predicting the persistence of human norovirus (NoV) seeded into surface water and groundwater used as source water for producing potable water is evaluated. Quantitative reverse transcription-PCR (qRT-PCR) assays are developed for each virus and the levels of infections virus and viral nucleic acid are monitored in spiked drinking source water over time.

An elution protocol was developed and combined with a rapid and sensitive real-time reverse transcription-PCR-based method to quantify adsorbed norovirus and rotavirus on bottle walls. The results strongly support the theory that bottled water is not a vehicle for viral infections.

A culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of [F.sup.+]-specific RNA coliphages using novel primer sets and molecular beacons is presented.

Freshwater and seawater samples were tested and compared by a modified real time quantitative reverse transcriptase (qRT-PCR) parameters, the cellulose mixed-ester filter retention with the retention of more porous and freely flowing glass fiber filters. It shows a readily usable means to detect human enteroviruses in field samples from freshwater and marine environments.

A gene-designated trt is discovered from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The conclusion states that the trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75 degree Celcius.

The development of an reverse transcription(RT)-booster PCR for the detection of Noroviruses ( NVs) both in shellfish and clinical samples is discussed. The reverse transcription-PCR method demonstrated an increase in sensitivity at least 2 log units for all the NV strains examined compared with the standard RT-PCR method and higher sensitivity was confirmed in tests on experimentally and naturally contaminated shellfish.