Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants.

The Rhizobium meliloti bacA gene encodes a function that is essential for bacterial differentiation into bacteroids within plant cells in the symbiosis between R. meliloti and alfalfa. An Escherichia coli homolog of BacA, SbmA, is implicated in the uptake of microcin B17, microcin J25 (formerly microcin 25), and bleomycin. When expressed in E. coli with the lacZ promoter, the R. meliloti bacA gene was found to suppress all the known defects of E. coli sbmA mutants, namely, increased resistance to microcin B17, microcin J25, and bleomycin, demonstrating the functional similarity between the two proteins. The R. meliloti bacA386::Tn(pho)A mutant, as well as a newly constructed bacA deletion mutant, was found to show increased resistance to bleomycin. However, it also showed increased resistance to certain aminoglycosides and increased sensitivity to ethanol and detergents, suggesting that the loss of bacA function causes some defect in membrane integrity. The E. coli sbmA gene suppressed all these bacA mutant phenotypes as well as the Fix- phenotype when placed under control of the bacA promoter. Taken together, these results strongly suggest that the BacA and SbmA proteins are functionally similar and thus provide support for our previous hypothesis that BacA may be required for uptake of some compound that plays an important role in bacteroid development. However, the additional phenotypes of bacA mutants identified in this study suggest the alternative possibility that BacA may be needed for membrane integrity, which is likely to be critically important during the early stages of bacterial differentiation within plant cells.     

Deficiency of a Sinorhizobium meliloti BacA mutant in alfalfa symbiosis correlates with alteration of the cell envelope

The BacA protein is essential for the long-term survival of Sinorhizobium meliloti and Brucella abortus within acidic compartments in plant and animal cells, respectively. Since both the S. meliloti and B. abortus bacA mutants have an increased resistance to bleomycin, it was hypothesized that BacA was a transporter of bleomycin and bleomycin-like compounds into the bacterial cell. However, our finding that the S. meliloti bacA mutant also has an increased sensitivity to detergents, a hydrophobic dye, ethanol, and acid pH supported a model in which BacA function affects the bacterial cell envelope. In addition, an S. meliloti lpsB mutant that is defective at a stage in infection of the host similar to that found for a bacA mutant is also sensitive to the same agents, and the carbohydrate content of its lipopolysaccharide (LPS) is altered. However, analysis of crude preparations of the bacA mutant LPS suggested that, unlike that for LpsB, BacA function did not affect the carbohydrate composition of the LPS. Rather, we found that at least one function of BacA is to affect the distribution of LPS fatty acids, including a very-long-chain fatty acid thought to be unique to the alpha-proteobacteria, including B. abortus.     

Nitrogen fixation mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.     

Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain

The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.     

Disruption of the <Emphasis Type="Italic"> Saccharomyces cerevisiae </Emphasis>cell-wall pathway gene <Emphasis Type="Italic"> SLG1</Emphasis> causes hypersensitivity to the antitumor drug bleomycin

Bleomycin is an antitumor drug that damages DNA via a free radical-dependent mechanism, and yeast mutants defective in DNA repair are hypersensitive to the drug. To identify possible pathways that may contribute to bleomycin resistance in yeast, we characterized a panel of bleomycin-sensitive mutants that were previously isolated by insertion mutagenesis using the transposon miniTn3::Leu2::LacZ::AMP( R). One of these mutants harbored a single insertion in the SLG1 gene, which encodes a cell membrane protein that senses cell wall stress, and functions to maintain cell wall function by activating the protein kinase C signaling pathway. Deletion of the SLG1 gene in parental strains caused hypersensitivity to bleomycin, and this correlated with an accumulation of damaged DNA. A plasmid that expresses the native SLG1 gene or that increases PKC1 gene dosage restored bleomycin resistance to the slg1Delta mutant. Two-dimensional gel electrophoresis revealed that exposure to bleomycin triggered the expression of certain proteins, presumably to maintain cell wall function, in a Slg1-dependent manner. In addition, mutants lacking cell wall function were found to be hypersensitive to bleomycin. We conclude that mutants deficient in proteins that maintain cell wall function are severely compromised in their ability to limit bleomycin entry into the cell. Therefore, these mutants are burdened with increased genotoxicity upon exposure to bleomycin in the medium. Our results show that major mechanisms other than DNA repair are operating in yeast to mediate bleomycin resistance.     

Cellular resistance to bleomycin in Saccharomyces cerevisiae is not affected by changes in bleomycin hydrolase levels.

Bleomycin is a glycopeptide drug that exerts potent genotoxic potential and is highly effective in the treatment of certain cancers when used in combination therapy. Unfortunately, however, tumors often develop resistance against bleomycin, and the mechanism of this resistance remains unclear. It has been postulated that bleomycin hydrolase, a protease encoded by the BLH1 gene in humans, may account for tumor resistance to bleomycin. In support of such a notion, earlier studies showed that exogenous expression of yeast Blh1 in human cells can enhance resistance to bleomycin. Here we show that (i) yeast blh1delta mutants are not sensitive to bleomycin, (ii) bleomycin-hypersensitive yeast mutants were no more sensitive to this agent upon deletion of the BLH1/LAP3/GAL6 gene, and (iii) overproduction of Blhl in either the parent or bleomycin-hypersensitive mutants did not confer additional resistance to these strains. Therefore, yeast Blh1 apparently has no direct role in protecting this organism from the lethal effects of bleomycin, even though the enzyme can degrade the drug in vitro. Clearly, additional studies are required to establish the actual biological role of Blh1 in yeast.     

Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106-30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant delta bacA,delta ybjG,delta pgpB in which the expression of bacA is impaired at 42 degrees C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.     

Bleomycin and X-ray-hypersensitive Chinese hamster ovary cell mutants: genetic analysis and cross-resistance to neocarzinostatin

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304-5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560-1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin. Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313-319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.     

Genetic analysis of the Sinorhizobium meliloti BacA protein: differential effects of mutations on phenotypes

Sinorhizobium meliloti strains lacking BacA function are impaired in symbiosis with alfalfa host plants and display altered sensitivities to a number of compounds relative to wild-type strains. With the goal of finding clues to the currently unknown biological function(s) of BacA, we carried out a genetic analysis to determine which amino acids are critical for protein function and to attempt to ascertain whether the multiple phenotypes that result from a bacA-null allele were the result of a common cause or whether BacA has multiple functions. We have created a set of 20 site-directed mutants in which selected individual amino acids in bacA were replaced with glycine residues. The resulting mutants were characterized to determine how the various amino acid changes affected a number of phenotypes associated with loss of BacA function. Mutants H165G, W182G, D198G, and R284G had null phenotypes for all functions assayed, while mutants W57G, S83G, S231G, and K350G were indistinguishable from wild-type strains. The remaining 12 site-directed mutants demonstrate mixed phenotypic characteristics and fall into a number of distinctly different groups. These observations may be consistent with a role for BacA in multiple, nonoverlapping functions.     

The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity

The bacA gene, the overexpression of which results in bacitracin resistance, was inactivated and shown to be non-essential for growth of Escherichia coli. It was proposed earlier that the bacA gene product may confer resistance to the antibiotic by phosphorylation of undecaprenol (Cain, B. D., Norton, P. J., Eubanks, W., Nick, H. S., and Allen, C. M. (1983) J. Bacteriol. 175, 3784-3789). In the present work, this extremely hydrophobic membrane protein was overproduced and purified to near homogeneity. The analysis of its catalytic properties clearly demonstrated that the purified BacA protein exhibited undecaprenyl pyrophosphate phosphatase activity but not undecaprenol phosphokinase activity. This finding was perfectly consistent with the mechanism of action of bacitracin that consists in the sequestration of undecaprenyl pyrophosphate, the BacA enzyme substrate. The level of undecaprenyl pyrophosphate phosphatase was increased by 280-fold in cells carrying bacA on a multicopy expression plasmid. It was decreased by approximately 75% but was not completely abolished in a bacA disruption mutant, suggesting that BacA is the main E. coli undecaprenyl pyrophosphate phosphatase but that other protein(s) exhibiting such an activity should exist to account for the residual activity and viability of the mutant strain. This is the first gene encoding undecaprenyl pyrophosphate phosphatase identified to date. Considering its newly identified function, we propose to rename the bacA gene uppP.     

Sequential expression of two late nodulin genes in the infected cells of alfalfa root nodules.

The Nms-22 and leghemoglobin (Lb) genes are expressed exclusively in the infected cells of alfalfa root nodules. Expression of these two late nodulin genes originated at distinct cellular boundaries within the symbiotic region of the nodule. The Nms-22 gene was expressed in all infected cells, including those just adjacent to the meristematic region. Lb gene expression was induced in older infected cells and was most prominent in the mature region of the nodule. Despite this temporal separation of gene expression, both the Nms-22 and Lb genes were expressed in nodules elicited by bacA mutants in which bacteroid development has been blocked just after release from the infection thread.     

Mutagenesis in Streptomycete cultures exposed to bleomycin

Mutagenic properties of bleomycin, an antitumor antibiotic were studied with respect to 2 species of streptomycetes producing practically important antibiotics. A multifold increase in the frequency of prototrophic revertants among the survivors of strains His- and Met- of Actinomadura carminata exposed to bleomycin was observed. Bleomycin was effective in induction of various morphological mutants, and auxotrophs at a high survival rate of the spores of Str. cremeus var. tobramycini, a tobramycin-producing organism. It was shown with the method of subsequent mutagenesis that the efficacy of induction of morphological and auxotrophic mutants in germinating spores of Actinomadura carminata, a carminomycin-producing organism by bleomycin in a concentration of 100 micrograms/ml and an exposure time of 5 minutes was much higher that in the latent spores. The mutagenic effect of bleomycin is comparable with that of ionizing radiation.     

Genetic analysis of Rhizobium meliloti bacA-phoA fusion results in identification of degP: two loci required for symbiosis are closely linked to degP.

The function of the Rhizobium meliloti bacA gene, which is a homolog of the Escherichia coli sbmA gene, is required for an intermediate step in nodule development. A strain carrying the bacA386::TnphoA fusion was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and three mutants that had higher levels of alkaline phosphatase activity were identified. The mutations in these strains were recessive and mapped to the same genetic locus. The gene affected by these mutations was identified and sequenced and was found to be a homolog of the E. coli degP gene, which encodes a periplasmic endopeptidase. Although degP function is important for the virulence of certain intracellular pathogens of mammals, it is not required for the R. meliloti-alfalfa symbiosis. The genetic analyses involving degP were complicated by the presence of a locus immediately upstream of depP that was lethal when present in multiple copies in a DegP- background. R. meliloti derivatives carrying insertion mutations in this locus displayed an N,N,N',N'-tetramethyl-p-phenylenediamine oxidase-negative phenotype, elicited the formation of white cylindrical nodules that did not fix nitrogen, and grew slowly in rich medium, suggesting that the locus was a cyc gene encoding a protein involved in the biosynthesis of a component or components of a respiratory chain. The previously identified fix-382::TnphoA, which similarly causes the formation of white cylindrical nodules that do not fix nitrogen, was shown to affect a gene that is separate from this cyc gene but extremely closely linked to it.     

Amplification of the bacA gene confers bacitracin resistance to Escherichia coli.

An Escherichia coli genomic library was constructed in order to facilitate selection for genes which confer bacitracin resistance through amplification. One of the plasmids from the library, plasmid pXV62, provided a high level of bacitracin resistance for E. coli. Deletion and nucleotide sequence analyses of bacitracin resistance plasmid pXV62 revealed that a single open reading frame, designated the bacA gene, was sufficient for antibiotic resistance. The bacA gene mapped to approximately 67 min on the E. coli chromosome by proximity to a previously mapped locus. The deduced amino acid sequence of the bacA-encoded protein suggests an extremely hydrophobic protein of 151 amino acids, approximately 65% of which were nonpolar amino acids. E. coli cells containing plasmid pXV62 have increased isoprenol kinase activity. The physical characteristics of the deduced protein and enhanced lipid kinase activity suggest that the bacA gene may confer resistance to bacitracin by phosphorylation of undecaprenol.     

Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways.

In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and RAD6-dependent DNA repair. Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration. Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin. The most important mechanisms involved are recombination and postreplication repair. The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the RAD6- and RAD52-dependent repair processes. Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations. RAD3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin. The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates. On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.     

RecBC promoted repair of bleomycin damage in Escherichia coli.

The repair response of Escherichia coli K-12 to bleomycin was examined in Rec- mutants showing differential sensitivity to this agent. Sedimentation analysis of the cellular DNA showed incision after bleomycin treatment. The subsequent reformation of the DNA, found in the wild-type and the recD mutant, was abolished in the recB and delayed in the recF and recBC sbcB mutants. The bleomycin-induced SOS response was reduced in strains containing recB or recBC sbsB mutations. It is suggested that the RecBCD pathway has the main role in the efficient repair of bleomycin-induced DNA damage.     

Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1.

The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bac1, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bac1 ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bac1, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broad-spectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.     

Endonuclease IV (nfo) mutant of Escherichia coli.

A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.