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In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis. Olga N. Sekurova, 2004.Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455 . Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficientnystatin production, whereas no significant roles could be demonstratedfor the other two regulatory genes . To determine the in vivotargets for the NysR regulators, chromosomal integration vectorswith the xylE reporter gene under the control of seven putativepromoter regions upstream of the nystatin structural and regulatorygenes were constructed . Expression analyses of the resultingvectors in the S . noursei wild-type strain and regulatory mutantsrevealed that the four regulators differentially affect certainpromoters . According to these analyses, genes responsible forinitiation of nystatin biosynthesis and antibiotic transportwere the major targets for regulation . Data from cross-complementationexperiments showed that nysR genes could in some cases substitutefor each other, suggesting a functional hierarchy of the regulatorsand implying a cascade-like mechanism of regulation of nystatinbiosynthesis. Bacillus subtilis YdiH Is a Direct Negative Regulator of the cydABCD Operon. Matthew Schau, 2004.During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa3, caa3, and bd . Cytochrome bd is encoded by the cydABCD operon . We report here the first identification of a regulator for the cydABCD operon, YdiH . While working with  resDE
mutant strains, we identified colonies which contained suppressor
mutations (cmp) which bypassed the requirement for ResD for
all phenotypes not associated with cytochrome aa3
or caa3 . Mapping identified a class of Tn10 insertions
which were close to the cmp locus (Tn10-2) and a
second class (Tn10-1) which was inserted in cydD,
a gene which appears to be essential to the cmp phenotype .
Sequencing of the cmp loci from four independent
resDE
cmp isolates yielded four loss-of-function alleles of ydiH,
a gene encoding a protein with homology to AT-rich DNA-binding
proteins . Additionally, we determined that cytochrome bd was
aberrantly expressed in the
resDE
cmp background . Together these data led to the hypothesis that
YdiH serves as a negative regulator of cydABCD expression, a
hypothesis supported by both gel-shift and DNase I footprinting
analyses . YdiH protected the cydA promoter region at three
22-bp repeats located in the long 5' untranslated region (193 bp) .
Induction of the cydABCD operon in a
resDE
background showed that expression of the terminal oxidase bd
was responsible for the bypass phenotype observed in a
resDE
cmp strain, indicating that cytochrome bd expression
complemented the loss of cytochromes aa3 and caa3
in the
resDE
strain .
Use of Sinorhizobium meliloti as an Indicator for Specific Detection of Long-Chain N-Acyl Homoserine Lactones. Inmaculada Llamas, 2004.Population-density-dependent gene expression in gram-negative bacteria involves the production of signal molecules characterized as N-acyl homoserine lactones (AHLs) . The synthesis of AHLs by numerous microorganisms has been identified by using biosensor strains based on the Agrobacterium tumefaciens and Chromobacterium violaceum quorum-sensing systems . The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti is rapidly becoming a model organism for the study of quorum sensing . This organism harbors at least three different quorum-sensing systems (Sin, Mel, and Tra), which play a role in its symbiotic relationship with its host plant, alfalfa . The Sin system is distinguished among them for the production of long-chain AHLs, including C18-HL, the longest AHL reported so far . In this work, we show that construction of a sinI::lacZ transcriptional fusion results in a strain that detects long-chain AHLs with exquisite sensitivity . Overexpression of the SinR regulator protein from a vector promoter increases its sensitivity without loss of specificity . We also show that the resulting indicator strain can recognize long-chain AHLs produced by unrelated bacteria such as Paracoccus denitrificans and Rhodobacter capsulatus . This S . meliloti indicator strain should serve as a tool for the specific detection of long-chain AHLs in new systems . SitABCD Is the Alkaline Mn2+ Transporter of Salmonella enterica Serovar Typhimurium. David G. Kehres, 2002.MntH, a bacterial homolog of the mammalian natural resistance-associated macrophage protein 1 (Nramp1), is a primary Mn2+ transporter of Salmonella enterica serovar Typhimurium and Escherichia coli . S . enterica serovar Typhimurium MntH expression is important for full virulence; however, strains carrying an mntH deletion are only partially attenuated and display no obvious signs of Mn2+ deficiency . We noted that promoter sequences for mntH and for the putative Fe2+ transporter sitABCD appeared to have the same regulatory element responsive to Mn2+ and so hypothesized that sitABCD could transport Mn2+ with high affinity . We have now characterized transport by SitABCD in S . enterica serovar Typhimurium using 54Mn2+ and 55Fe2+ and compared its properties to those of MntH . SitABCD mediates the influx of Mn2+ with an apparent affinity (Ka) identical to that of MntH, 0.1 µM . It also transports Fe2+ but with a Ka 30 to 100 times lower, 3 to 10 µM . Inhibition of 54Mn2+ transport by Fe2+ and of 55Fe2+ transport by Mn2+ gave inhibition constants comparable to each cation's Ka for influx . Since micromolar concentrations of free Fe2+ are improbable in a biological system, we conclude that SitABCD functions physiologically as a Mn2+ transporter . The cation inhibition profiles of SitABCD and MntH are surprisingly similar for two structurally and energetically unrelated transporters, with a Cd2+ Ki of  1 µM and a Co2+ Ki of
20 µM and with Ni2+, Cu2+, and Fe3+ inhibiting both transporters only at concentrations of >0.1 mM . The one difference is that Zn2+ exhibits potent inhibition of SitABCD (Ki = 1 to 3 µM) but inhibits MntH weakly (Ki > 50 µM) . We have previously shown that MntH transports Mn2+ most effectively under acidic conditions . In sharp contrast, SitABCD has almost no transport capacity at acid pHs and optimally transports Mn2+ at slightly alkaline pHs . Overall, coupled with evidence that each transporter is multiply but distinctly regulated at the transcriptional level, the distinct transport properties of MntH versus SitABCD suggest that each transporter may be specialized for Mn2+ uptake in different physiological environments .
Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying Pseudomonas sp . Strain FA1. Bharat Bhushan, 2003.The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood . Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment . In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp . strain FA1, was isolated from a garden soil . Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation . An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells . The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h-1 mg of cell biomass-1 and 11.5 ± 0.4 nmol h-1 mg of protein-1, respectively, under anaerobic conditions . In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO2-), 1.5 molecules of nitrous oxide (N2O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule . The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions . A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme . The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20 .
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