Inhalation of benzene leads to an increase in the mutant frequencies of a lacI transgene in lung and spleen tissues of mice.

The goal of this study was to determine if inhalation of benzene leads to an increase in the mutant frequencies in the tissues of male C57BL/6 mice. Mutant frequencies were measured using a previously described assay in which bacteriophage lambda lacI transgenes are rescued from mouse genomic DNA as infectious phage and scored for their LacI phenotype. Eight experimental mice were exposed to a target concentration of 300 ppm of benzene for 6 h/day x 5 days/week x 12 weeks, and eight control mice were treated similarly except that they were not exposed to benzene. Mutant frequencies were calculated as the ratio of LacI-/total phage recovered from organs of interest. The mean mutant frequency measured in lung tissues of mice exposed to benzene was (10.6 +/- 1.4) x 10(-5), which is about 1.7-fold higher than that of the unexposed controls. In spleen tissues from benzene-exposed mice the mean mutation frequency was (12.6 +/- 4.1) x 10(-5), which is about 1.5-fold higher than that of spleen tissues from unexposed controls. The differences in mean mutant frequencies between benzene-exposed and unexposed lung and spleen tissues are statistically significant. In liver tissues, however, the mean mutant frequencies of benzene-exposed mice and unexposed mice are not significantly different. These results demonstrate that inhaled benzene results in a statistically significant increase in the mutant frequencies in lung and spleen, but not in liver tissues of mice.

Cis- and trans-acting elements required for regulation of flagellar gene transcription in the bacterium Caulobacter crescentus.

The flagellar genes of the bacterium Caulobacter crescentus are organized into a regulatory hierarchy that consists of four classes or levels of genes, and expression of these genes is restricted to a discrete interval in the cell cycle that begins just prior to flagellum assembly. This paper summarizes data on the promoters and other cis-acting elements that are required for transcription of the level II gene fliF and the level III genes flaN and flbG. Regulated expression of flaN and flbG requires sigma 54 promoters, enhancer-like sequences called ftr, and sequences called ihf that conform to the consensus binding sequence for Escherichia coli integration host factor protein. The fliF regulatory region contains a new type of promoter sequence that is different from other known promoter motifs, and it has a sequence called ftr4 that is a site of negative regulation. ftr4 appears to function as part of a developmental switch that turns fliF transcription off at the correct time in the cell cycle. flbD, the last gene in the fliF operon is a negative regulator of fliF and an activator of both flaN and flbG expression. Evidence that FlbD protein plays a direct role as a transcriptional regulator comes from the finding that it has a DNA binding activity within its carboxy terminus that specifically recognizes ftr4 in fliF and four ftr elements in the flaN-flbG promoter region.

Genetic organization and transposition properties of IS511.

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobacter crescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISR1, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5' end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.

FlbD has a DNA-binding activity near its carboxy terminus that recognizes ftr sequences involved in positive and negative regulation of flagellar gene transcription in Caulobacter crescentus.

FlbD is a transcriptional regulatory protein that negatively autoregulates fliF, and it is required for expression of other Caulobacter crescentus flagellar genes, including flaN and flbG. In this report we have investigated the interaction between carboxy-terminal fragments of FlbD protein and enhancer-like ftr sequences in the promoter regions of fliF, flaN, and flbG. FlbDc87 is a glutathione S-transferase (GST)-FlbD fusion protein that carries the carboxy-terminal 87 amino acids of FlbD, and FlbDc87 binds to restriction fragments containing the promoter regions of fliF, flaN, and flbG, whereas a GST-FlbD fusion protein carrying the last 48 amino acids of FlbD failed to bind to these promoter regions. DNA footprint analysis demonstrated that FlbDc87 is a sequence-specific DNA-binding protein that makes close contact with 11 nucleotides in ftr4, and 6 of these nucleotides were shown previously to function in negative regulation of fliF transcription in vivo (S. M. Van Way, A. Newton, A. H. Mullin, and D. A. Mullin, J. Bacteriol. 175:367-376, 1993). Three DNA fragments, each carrying an ftr4 mutation that resulted in elevated fliF transcript levels in vivo, were defective in binding to FlbDc87 in vitro. We also found that a missense mutation in the recognition helix of the putative helix-turn-helix DNA-binding motif of FlbDc87 resulted in defective binding to ftr4 in vitro. These data suggest that the binding of FlbDc87 to ftr4 is relevant to negative transcriptional regulation of fliF and that FlbD functions directly as a repressor. Footprint analysis showed that FlbDc87 also makes close contacts with specific nucleotides in ftr1, ftr2, and ftr3 in the flaN-flbG promoter region, and some of these nucleotides were shown previously to be required for regulated transcription of flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 175:2067-2076, 1993). Footprint analysis also revealed a new ftr-like sequence, ftr5, at -136 from the transcription start site of flbG. Our results demonstrate that FlbD contains a sequence-specific DNA-binding activity within the 87 amino acids at its carboxy terminus, and the results suggest that FlbD exerts its effect as a positive and negative regulator of C. crescentus flagellar genes by binding to ftr sequences.

Inhaled benzene increases the frequency and length of lacI deletion mutations in lung tissues of mice.

This study investigated the frequency and pattern of mutations that arose in lacI transgenes in lung tissues of mice exposed to 300 p.p.m. of benzene for 6 h/day x 5 days/week for 12 weeks. The nucleotide sequence changes in 86 lacI- transgenes from lung tissues of eight benzene-exposed mice (BEM) and 78 spontaneous lacI- transgenes from lung tissues of eight unexposed control mice (UCM) were identified and compared. A total of 31% (27/86) of the lacI mutations in BEM are deletions compared with 9% (7/78) deletions in UCM. In BEM, 44% (12/27) of the deletions were longer than 10 bp, whereas only 14% (1/7) of the deletions in UCM exceeded 10 bp in length. Statistical tests supported the hypothesis that benzene exposure resulted in significant increases in both the frequency and length of deletions. Based on the lacI mutant frequency and fraction of unique mutations, lung tissues of BEM were estimated to have a 1.8-fold increase in lacI mutation frequency compared with lung tissues of UCM. The results presented in this paper demonstrate that inhaled benzene is a gene mutagen in lung tissues of mice.

Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar hook.

This paper reports on the organization, expression, and function of the divergently transcribed flbG and flaN operons in the hook gene cluster of Caulobacter crescentus. The transcription initiation site of flbG was determined previously, and in this work the transcription map was completed by locating the 3' end of the mRNA using nuclease S1 protection assays. A previous genetic study had suggested that the flbG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5'-flbG, flbH, and flgE. FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaffolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein. The divergently transcribed flaN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins. Based on the amino acid sequence similarities and phenotypes of mutants, flbG, flbH, and flaN have been renamed fliK, flgD, and flgK, FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identified as part of a 190-kDa FliK-LacZ fusion protein. We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly.

Identification of the promoter and a negative regulatory element, ftr4, that is needed for cell cycle timing of fliF operon expression in Caulobacter crescentus.

The fliF operon of Caulobacter crescentus, which was previously designated the flaO locus, is near the top of the flagellar-gene regulatory hierarchy, and it is one of the earliest transcription units to be expressed in the cell cycle. In this report, we have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription. The first sequence was defined by the effects of three 2-bp deletions and five point mutations, each of which greatly reduced the level of fliF operon transcript in vivo. These eight mutations lie between -37 and -22 within an 18-bp sequence that matches, at 11 nucleotides, sequences in the 5' regions of the flaQR (flaS locus) and fliLM operons, which are also expressed early and occupy a high level in the regulatory hierarchy (A. Dingwall, A. Zhuang, K. Quon, and L. Shapiro, J. Bacteriol. 174:1760-1768, 1992). We propose that this 18-bp sequence contains all or part of the fliF promoter. We have also identified a second sequence, 17 bp long and centered at -8, which we have provisionally designated ftr4 because of its similarity to the enhancer-like ftr sequences required for regulation of sigma 54 promoters flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). Six of the seven mutations in ftr4 examined resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation. A 2-bp deletion at -12 and -13 in ftr4 altered the cell cycle pattern of fliF operon transcription; the transcript was still expressed periodically, but the period of its synthesis was extended significantly. We suggest that the ftr4 sequence may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle.