bacA Gene Research Articles

The effects of food provisioning on the gut microbiota community and antibiotic resistance genes of Yunnan snub-nosed monkey.

Yunnan snub-nosed monkeys (Rhinopithecus bieti) are the highest elevation lived non-human primate, and their survival has been threatened for decades. To promote their population growth, a reserve provides a typical monkey population with supplemental food. However, the influences of this food provisioning on their gut microbiota and antibiotic resistance genes (ARGs) were unknown. Therefore, we investigated the gut microbiota and ARGs of the food-provisioned monkey population compared with another wild foraging population. We found that food provisioning significantly increased the gut microbiota diversity and changed the community composition, particularly increased both the Firmicutes abundance and Firmicutes/Bacteroidetes ratio. Meanwhile, the food provisioning decreased the complex and stable gut microbiota network. KEGG functions were also influenced by food provisioning, with wild foraging monkeys showing higher functions of metabolism and genetic information processing, especially the carbohydrate metabolism, while food-provisioned monkeys exhibited increased environmental information processing, cellular processes, and organismal systems, including valine, leucine, and isoleucine degradation. In addition, food provisioning increased the abundance of ARGs in the gut microbiota, with most increasing the abundance of bacA gene and changing the correlations between specific ARGs and bacterial phyla in each population. Our study highlights that even food provisioning could promote wildlife nutrient intake, and it is necessary to pay attention to the increased ARGs and potential effects on gut microbiota stability and functions for this human conservation measure.

Immunogenicity and efficacy of an oral live-attenuated vaccine for bovine Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of Johne's disease (JD) in ruminants, establishes a prolonged and often lifelong enteric infection. The implementation of control measures for bovine JD has faced obstacles due to the considerable expenses involved in disease surveillance and hindered by unreliable and inadequate diagnostic tests, emphasizing the need for an effective vaccine that can stimulate mucosal immunity in the gastrointestinal tract. Previous investigations have demonstrated that deletion of the BacA gene in MAP produces an attenuated strain that can transiently colonize the calf small intestine while retaining its capacity to stimulate systemic immune responses similar to wildtype MAP strains. This study assessed the efficacy of the BacA gene deletion MAP strain, referred to as the BacA vaccine, when administered orally to young calves. The research aimed to evaluate its effectiveness in controlling MAP intestinal infection and to investigate the immune responses elicited by mucosal vaccination. The study represents the first evaluation of an enteric modified live MAP vaccine in the context of an oral MAP challenge in young calves. Oral immunization with BacA reduced MAP colonization specifically in the ileum and ileocecal valve. This partially protective immune response was associated with an increased frequency of CD4+ and CD8+ T cells with a pro-inflammatory phenotype (IFNγ+/TNFα+) in vaccinated animals. Moreover, re-stimulated PBMCs from vaccinated animals showed increased expression of IFNγ, IP-10, IL-2, and IL-17 at 10- and 12-weeks post challenge. Furthermore, immunophenotyping of blood leukocytes revealed that vaccinated calves had increased levels of T cells expressing cell-surface markers consistent with long-term central memory. Overall, our findings provide new insights into the development and immunogenicity of a modified live MAP vaccine against bovine JD, demonstrating oral vaccination can stimulate host immune responses that can be protective against enteric MAP infection.

Anthropogenic disturbances on antibiotic resistome along the Yarlung Tsangpo River on the Tibetan Plateau: Ecological dissemination mechanisms of antibiotic resistance genes to bacterial pathogens

Human activities can accelerate the antibiotic resistome prevalence and pose threats to ecological safety and public health globally. However, antibiotic resistance gene (ARG) mobility and dissemination into bacterial pathogens under anthropogenic disturbances are still poorly understood. Here, we used a metagenomic approach to profile the biogeography of ARGs and pathogenic antibiotic resistant bacteria (PARB) under anthropogenic disturbances along the Yarlung Tsangpo River. Results showed the ARGs was dominated by bacA gene along the Yarlung Tsangpo River on the Tibetan Plateau. The ARG composition was differently impacted by rapid urbanization and dam construction, which urbanization could promote ARGs resistant to sulfonamide and tetracycline, whereas dam construction could elevate the resistance to chloramphenicol and aminoglycoside. Land use pattern was identified as a critical factor influencing ARG composition under anthropogenic disturbances, as it could directly reflect the land degradation level and indicate the inputs of ARG-selective chemicals of different human activities. Moreover, despite of the lack of variation in ARG relative abundance, PARB were highly promoted by anthropogenic activities, indicating increasing ARG dissemination to pathogen. We found that human-impacted environments harbored high proportion of mobile genetic elements (MGEs), and the MGE carrying ARGs also increased under anthropogenic disturbances in the pathogenic hosts, which confirmed that anthropogenic activities could promote ARG horizontal gene transfer. Furthermore, anthropogenic activities could influence PARB assembly processes. Basically, stochastic processes dominated PARB assembly along the river, and with increasing level of anthropogenic activities, these processes shifted from undominated stochastic processes to dispersal limitation. In summary, this study provides useful strategies in watershed resistome management and reduction of ARG dissemination to pathogens, which should consider the mode and intensity of human activity and its potential influence on horizontal gene transfer and assembly processes.

Identification and characterization of antimicrobial peptide produced by indigenously isolated Bacillus paralicheniformis UBBLi30 strain.

The antimicrobial compound produced by Bacillus paralicheniformis UBBLi30 showed UV spectra absorption at 208nm. Fourier transform infrared (FTIR) revealed characteristic bands for aliphatic chain related to hydrophobic amino acids (l-isoleucine/l-leucine) (3068, 2965 and 2871cm-1) and peptide bonds (1538, 1667 and 3312cm-1). The proton nuclear magnetic resonance (1H NMR) showed signals for aromatic amino acid (6.5-9.5ppm) and alkyl amines (3-4ppm). The results of carbon (13C) NMR showed signals for aromatic, nitro and amide compounds. Besides this, the mass fragments (1422.576 [M+H]+, 711.912 [M+2H]2+ and 475.174 [M+3H]3+m/z) observed in electrospray ionization mass spectrometry (ESI-MS) were coordinated well to the fragments of polypeptide antibiotic bacitracin. The presence of bacA gene further confirmed the production of bacitracin. Bacitracin inhibited the growth of a range of Gram-positive bacteria such as Micrococcus luteus, methicillin-resistant Staphylococcus aureus (MRSA), S. aureus, Streptococcus pyogenes and Propionibacterium acnes, and biofilm formation of M. luteus and MRSA. Moreover, this polypeptide reduced the zeta potential of M. luteus and MRSA, indicating the electrostatic sorption on bacterial surface and concentration-dependent cell membrane damages. Besides this, polypeptide showed stability in the presence of proteases (proteinase K, trypsin and pepsin), pH (1, 3, 5, 7, 9 and 11) and temperature up to 100°C. B. paralicheniformis UBBLi30 therefore has the potential to be utilized as a bio-preservative to control the growth of spoilage and pathogenic bacteria.

Heterologous Complementation Reveals a Specialized Activity for BacA in the Medicago-Sinorhizobium meliloti Symbiosis.

The bacterium Sinorhizobium meliloti Rm2011 forms N2-fixing root nodules on alfalfa and other leguminous plants. The pSymB chromid contains a 110-kb region (the ETR region) showing high synteny to a chromosomally located region in Sinorhizobium fredii NGR234 and related rhizobia. We recently introduced the ETR region from S. fredii NGR234 into the S. meliloti chromosome. Here, we report that, unexpectedly, the S. fredii NGR234 ETR region did not complement deletion of the S. meliloti ETR region in symbiosis with Medicago sativa. This phenotype was due to the bacA gene of NGR234 not being functionally interchangeable with the S. meliloti bacA gene during M. sativa symbiosis. Further analysis revealed that, whereas bacA genes from S. fredii or Rhizobium leguminosarum bv. viciae 3841 failed to complement the Fix- phenotype of a S. meliloti bacA mutant with M. sativa, they allowed for further developmental progression prior to a loss of viability. In contrast, with Melilotus alba, bacA from S. fredii and R. leguminosarum supported N2 fixation by a S. meliloti bacA mutant. Additionally, the S. meliloti bacA gene can support N2 fixation of a R. leguminosarum bacA mutant during symbiosis with Pisum sativum. A phylogeny of BacA proteins illustrated that S. meliloti BacA has rapidly diverged from most rhizobia and has converged toward the sequence of pathogenic genera Brucella and Escherichia. These data suggest that the S. meliloti BacA has evolved toward a specific interaction with Medicago and highlights the limitations of using a single model system for the study of complex biological topics.

Characterization of StyR-234 non-coding RNA in regulation of Multi-drug resistance in Salmonella enterica serovar typhi

Non-protein coding RNAs (npcRNA) have been recently recognized in bacterial pathogens as a new group to regulate multi-drug resistance. Among several pathogenic bacterial spcecices, Salmonella enterica serovar Typhi (S. typhi) one of the focusing target in order to prevent severe impact in human health and it pose a problem by causing high fever. we report the characterization of StyR-234, a small antisense npcRNA, discovered from Salmonella typhi. StyR-234 is located on positive strand and antisense to, bacA gene. Since bacA is known to participate in MDR, we hypothesis StyR-234 is involve in bacA regulation thus could have role in drug resistance in S. typhi. Overexperssion analysis show that S. typhi became more susceptible to six antibiotics (Amikacin, Cefepime, Trimethoprim Sulphamethoxazole, Imipenem, Meropenem, Piperacillin Tazobactam) while more resistance to two antibiotics (Ampicillin and Cefotaxime). And no changes in sensitivity for three antibiotics (Ciprofloxacin, Clindamycin and Ceftazidime). The results indicated that StyR234 increasing susceptibility to many antibiotics could be by down regulating antibiotic resistant gene bacA.

How Symbiotic Bacteria Survive Host Defenses

There’s a surprisingly fine line between bacterial symbiosis and chronic infection. While one is beneficial and the other detrimental, recent findings suggest that they share mechanisms for sidestepping host defenses. Plants in the pea family (legumes) have symbiotic nitrogen-fixing bacteria living in root nodule compartments that also contain antimicrobial compounds. For example, the nitrogenfixer Sinorhizobium meliloti survives antimicrobial compounds called nodule-specific, cysteine-rich (NCR) peptides that are produced by alfalfa and related legumes. Intriguingly, S. meliloti is closely related to Brucella abortus, which causes abortions in cattle and can also cause debilitating chronic infections in people. Moreover, the host legume antimicrobial peptides that S. meliloti evades are similar to defensins, small proteins that help plants and animals kill bacteria. Once inside root nodule compartments, S. meliloti differentiates from the free-living form into elongated bacteroids that fix nitrogen. This differentiation is mediated by antimicrobial NCR peptides on the plant side, but the corresponding factors on the bacterial side are unknown. An S. meliloti cytoplasmic membrane protein called BacA has long been known to be essential for bacteroid development in alfalfa relatives, but its role has remained uncertain. Recently, however, BacA has been linked to NCR peptides: while BacA is required for S. meliloti differentiation in legumes that produce NCR peptides (such as alfalfa and peas), this protein is not required for bacteroid development in legumes that lack these peptides (such as beans and lotus). Now, in this issue of PLoS Biology, Mergaert, Ferguson, and colleagues present compelling evidence that BacA is key to protecting S. meliloti from antimicrobial NCR peptides produced by host legumes. To explore the role of BacA further, the researchers synthesized an antimicrobial peptide (NCR247) of the alfalfa relative Medicago truncatula and compared its effects on cultured S. meliloti with and without BacA. They found that while NCR247 decreases wild-type S. meliloti colony formation, this inhibition is more pronounced in a mutant strain that lacks BacA. In addition, this mutant’s growth was restored by a plasmid carrying the bacA gene. Taken together, these findings suggest that BacA protects cultured S. meliloti against NCR peptides. To test the effects of BacA in living plants, the researchers compared the viability of the wild-type and BacA-deficient S. meliloti strains. Nitrogen-fixing bacteria use infection threads to enter root nodule compartments, which contain NCR peptides that damage the cytoplasmic membrane of S. meliloti. Using a fluorescent dye test that stains dead bacteria red and stains living bacteria green, the researchers showed that both the wild-type and BacA-deficient S. meliloti were alive in the infection threads. In contrast, while the wild-type bacteria were still alive once inside the host root nodule compartments, most of the BacA-deficient mutants were dead. This suggests that without the protection of BacA, S. meliloti is rapidly killed by host antimicrobial NCR peptides in the compartments. To confirm the hypothesis that BacA protects S. meliloti against antimicrobial NCR peptides, the researchers also applied the fluorescent dye test to an M. truncatula mutant that blocks NCR peptide transport into root nodule compartments. As expected, both wild-type and BacAdeficient S. meliloti thrived within this mutant’s host compartments. Extending the similarities between the symbiotic S. meliloti and bacteria that cause chronic infections, BacA-like proteins are also found in B. abortus and Mycobacterium tuberculosis. In addition, the B. abortus BacA is essential for chronic infections in mammals. Previous work had shown that B. abortus BacA protects S. meliloti mutants that lack this protein against antimicrobial peptides in alfalfa relatives, and the researchers likewise found that B. abortus BacA protected these S. meliloti mutants against the synthesized antimicrobial peptide (NCR247) in culture. Altogether, these findings suggest a functional similarity between the S. meliloti and B. abortus BacA proteins, and show that the latter protects against antimicrobial activity like that of the defensins found in its mammalian hosts. This work suggests that by protecting S. meliloti against antimicrobial NCR peptides, BacA is key to establishing the symbiosis between these nitrogen-fixing bacteria and their legume hosts. How does BacA confer protection and limit the damage these compounds cause to the S. meliloti cytoplasmic membrane? Possibilities include a direct role for BacA in NCR transport or an indirect effect of BacA on outer membrane lipids. Because BacA is also key to establishing pathogenic bacteria including B. abortus in animal hosts, the answers could have widespread implications for understanding and ultimately treating such chronic infections in mammals.

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the ΔparA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the ΔparA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.

Streptomyces turgidiscabies Car8 contains a modular pathogenicity island that shares virulence genes with other actinobacterial plant pathogens

Streptomyces turgidiscabies Car8 is an actinobacterium that causes the economically important disease potato scab. Pathogenesis in this species is associated with a mobile pathogenicity island (PAISt) that site specifically inserts into the bacA gene in Streptomyces spp. Here we provide the 674,223 bp sequence of PAISt, which consists of two non-overlapping modules of 105,364 and 568,859 bp. These modules are delimited by three copies of an 8 bp palindromic sequence (TTCATGAA), that also is the integration site ( att) of the element. Putative tyrosine recombinase (IntSt) and excisionase (XisSt) proteins are encoded just upstream of att-R. PAISt has regions of synteny to pathogenic, symbiotic and saprophytic actinomycetes. The 105,364 bp PAISt module is identical to a genomic island in Streptomyces scabies 87–22, while the 568,859 bp module contains only a short region of synteny to that genome. However, both modules contain previously characterized and candidate virulence genes.

The bacA Gene Homolog, mlr7400, in Mesorhizobium loti MAFF303099 is Dispensable for Symbiosis with Lotus japonicus but Partially Capable of Supporting the Symbiotic Function of bacA in Sinorhizobium meliloti

Establishment of rhizobium-legume symbiosis requires a series of mutual authentication, which might involve bacterial evasion of host defense. One such evasion-related genes is Sinorhizobium meliloti bacA that is essential for bacteroid formation. BacA is a transmembrane protein highly similar to Escherichia coli SbmA, a predicted transporter, and has homologs even in animal pathogens, such as Brucella abortus in which the homolog contributes to effective survival in host macrophages. Despite such a significance in host-microbe interactions, studies on rhizobial BacA have been mostly performed with the Medicago-Sinorhizobium model system that forms indeterminate cylindrical nodules. Since Lotus japonicus-Mesorhizobium loti constitutes another model system that forms determinate globular nodules, we genetically analyzed the bacA homolog with the locus tag mlr7400 in M. loti MAFF303099. We found that the mlr7400-null mutant ML7400DK was able to establish quasi-healthy symbiosis with the Lotus plant with 50-80% nitrogen-fixing capacity. This dispensability for symbiosis was in contrast to the indispensability of S. meliloti BacA for symbiosis. However, free-living phenotypes of ML7400DK paralleled those of known bacA mutants, i.e. ML7400DK showed decreased sensitivity to the antibiotics bleomycin and gentamicin as well as increased sensitivity to membrane-disturbing reagents such as SDS. Conservation of the free-living function between Mlr7400 protein and S. meliloti BacA was further confirmed by heterologous complementation experiments. Although simple introduction of mlr7400 into the S. meliloti bacA mutant did not increase the symbiotic capacity at all, a significant but marginal increase was obtained when mlr7400 was fused to the S. meliloti bacA promoter. These findings might indicate currently progressing evolutionary specialization among BacA-SbmA proteins.

BacA is indispensable for successful Mesorhizobium–Astragalus symbiosis

A homologue of Sinorhizobium meliloti bacA was isolated from Mesorhizobium huakuii 7653R, which is capable of fixing atmospheric nitrogen in symbiotic association with leguminous Astragalus sinicus (Chinese milk vetch). Inactivation of the bacA gene abolished the ability of M. huakuii 7653R to establish a successful symbiosis with its host plant. Simultaneously, compared with wild-type M. huakuii 7653R, the bacA mutant was more sensitive to cell envelope-disrupting agents (acidic solution, ethanol, SDS, and crystal violet). Mass spectrometry analysis revealed that the very-long-chain fatty acid (27-OHC-28:0 and 29-OHC-30:0) contents of lipid A was reduced in the M. huakuii 7653R bacA mutant. Taken together, our data suggest that the cell envelope was altered in the M. huakuii 7653R bacA mutant, which might deteriorate bacterial adaption to acute environmental changes encountered in host cells and ultimately result in the failure of Mesorhizobium-legume symbiosis.

BacA, an ABC transporter involved in maintenance of chronic murine infections with Mycobacterium tuberculosis.

BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.

Cloning and characterization of a heat shock protein 70 gene, <italic>MsHSP70-1</italic>, in <italic>Medicago sativa</italic>

Some members of the heat shock protein 70 (HSP70) family have important functions in organism development. Here, we identified an alfalfa (Medicago sativa L. cv. Algonquin) HSP70 gene, MsHSP70-1, using cDNA array and reverse transcription-polymerase chain reaction. This gene contains a 1947 bp open reading frame encoding a protein of 649 amino acids. This protein contains all conserved domains, motifs, and characteristic sequences of plant HSP70s. The expression of MsHSP70-1 is enhanced in nodule compared with root, stem, leaf, and flower, and throughout the process of nodule development. Northern hybridization analysis indicated that the expression of MsHSP70-1 in nodule requires the active bacA gene of rhizobia. These results suggested that MsHSP70-1 might play an important role in alfalfa nodule development.

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.

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.

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.

Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis.

During the symbiosis between the bacterium Rhizobium meliloti and plants such as alfalfa, the bacteria elicit the formation of nodules on the roots of host plants. The bacteria infect the nodule, enter the cytoplasm of plant cells and differentiate into a distinct cell type called a bacteroid, which is capable of fixing atmospheric nitrogen. To discover bacterial genes involved in the infection and differentiation stages of symbiosis, we obtained genes expressed at the appropriate time and place in the nodule by identifying promoters that are able to direct expression of the bacA gene, which is required for bacteroid differentiation. We identified 230 fusions that are expressed predominantly in the nodule. Analysis of 23 sequences indicated that only three encode proteins known to be involved in the Rhizobium-legume symbiosis, six encode proteins with homology to proteins not previously associated with symbiosis, and 14 have no significant similarity to proteins of known function. Disruption of a locus that encodes a protein with homology to a cell adhesion molecule led to a defect in the formation of nitrogen-fixing nodules, resulting in an increased number of nitrogen-starved plants. Our isolation of a large number of nodule-expressed genes will help to open the intermediate stages of nodulation to molecular analysis.

The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases.

The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template. The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes. The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BA1, 598kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723kDa) with additional internal epimerization domains attached to the second and fourth. A carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant. The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.

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.

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.