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Previous studies have shown that a [[sigma].sup.54][[sigma].sup.s] cascade regulates the expression of a few key lipoproteins in Borrelia burgdorferi, the agent of Lyme disease. Here, we demonstrate that these sigma factors, both together and independently, regulate a much more extensive number of genes and cellular processes. Microarray analyses of [[sigma].sup.54] and [[sigma].sup.s] mutant strains identified 305 genes regulated by [[sigma].sup.54] and 145 regulated by [[sigma].sup.s] whereas the [[sigma].sup.54]-[[sigma].sup.s] regulatory cascade appears to control 48 genes in B. burgdorferi. In silico analyses revealed that nearly 80% of genes with altered expression in the [[sigma].sup.54] mutant were linked to potential [[sigma].sup.54]-dependent promoters. Many [[sigma].sup.54]-regulated genes are expressed in vivo, and through genetic complementation of the mutant, we demonstrated that [[sigma]sub.54] was required by B. burgdorferi to infect mammals. Surprisingly, [[sigma].sup.54] mutants were able to infect Ixodes scapularis ticks and be maintained for at least 24 wk after infection, suggesting the [[sigma].sup.54] [[sigma].sup.-s] regulatory network was not involved in long-term survival in ticks. However, [[sigma].sup.54] mutants did not enter the salivary glands during tick feeding, indicating that [[sigma].sup.54]-regulated genes were involved in the transmission process. infectivity | microarray | Lyme | transcription

Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g [cm.sup.-3]). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g [cm.sup.-3]) and low buoyant density (1.169 to 1.171 g [cm.sup.-3]). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g [cm.sup.-3]) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.

Research reveals that enzyme Gpm of the Embden-Meyerhof pathway is responsible for the heat sensitivity of Treponema pallidum. Data indicate that Gpm is stable for four hours at 25 C, losing 50% of the activity at 34 C and completely inactive at 42 C.

The purified membranes of Serpulina hyodysenteriae was examined to determine whether they contained cholesterol. The study made use of discontinuous sucrose density gradients to isolate outer and inner membranes. Results revealed that the outer and inner membrane fractions contained less than 1% and 2% of the total NADH oxidase activity in the cell lysate. Treatment with different concentrations of digitonin showed that the outer membrane could be selectively be removed at concentrations to about 0.125%.

The purification and characterization of an unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 revealed that its pH optimum is different from all other previously reported xylose isomerases. Its maximal activity is placed at pH 6.4 or pH 6.8 in a 30-minute assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. It also required Mn(super 2+), Co(super 2+) or Mg(super 2+) for thermal stability and isomerase activity and preferred xylose as a substrate.

Isopycnic centrifugation using sucrose density gradations isolates the inner and outer membranes of Borrelia burgdorferi and assists in its characterization. A soluble marker and an inner membrane marker help in the structural and component analysis of the outer membrane, which contains small quantities of flagellin protein, implying that it is almost pure. The number of intramembranous particles per square micrometer in the outer and inner membrane is 6,600 and 1,700 respectively, as revealed by freeze-fracture electron microscopy.

Summary: Polyamines are an essential class of metabolites found throughout all kingdoms in life. Borrelia burgdorferi harbors no enzymes to synthesize or degrade polyamines yet does contain a polyamine uptake system, potABCD. In this report, we describe the initial characterization of this putative transport system. After several unsuccessful attempts to inactivate potABCD, we placed the operon under the control of an inducible LacI promoter expression system. Analyses of this construct confirmed that potABCD was required for in vitro survival. Additionally, we demonstrated that the potABCD operon were upregulated in vitro by low osmolarity. Previously, we had shown that low osmolarity triggers the activation of the Rrp2/RpoN/RpoS regulatory cascade, which regulates genes essential for the transmission of spirochetes from ticks to mammalian hosts. Interestingly, induction of the pot operon was only affected in an rpoS mutant but not in a rpoN mutant, suggesting that the genes were RpoS dependent and RpoN independent. Furthermore, potABCD was upregulated during tick feeding concomitant with the initiation of spirochete replication. Finally, uptake experiments determined the specificity of B. burgdorferi's PotABCD for spermidine. [ABSTRACT FROM AUTHOR]