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.     

Importance of unusually modified lipid A in Sinorhizobium stress resistance and legume symbiosis

Sinorhizobium meliloti, a legume symbiont and Brucella abortus, a phylogenetically related mammalian pathogen, both require their BacA proteins to establish chronic intracellular infections in their respective hosts. The lipid A molecules of S. meliloti and B. abortus are unusually modified with a very-long-chain fatty acid (VLCFA; C > or = 28) and we discovered that BacA is involved in this unusual modification. This observation raised the possibility that the unusual lipid A modification could be crucial for the chronic infection of both S. meliloti and B. abortus. We investigated this by constructing and characterizing S. meliloti mutants in the lpxXL and acpXL genes, which encode an acyl transferase and acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A. Our analysis revealed that the unusually modified lipid A is important, but not crucial, for S. meliloti chronic infection and that BacA must have an additional function, which in combination with its observed effect on the lipid A in the free-living form of S. meliloti, is essential for the chronic infection. Additionally, we discovered that in the absence of VLCFAs, S. meliloti produces novel pentaacylated lipid A species, modified with unhydroxylated fatty acids, which are important for stress resistance.     

Role of BacA in lipopolysaccharide synthesis, peptide transport, and nodulation by Rhizobium sp. strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.     

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.     

Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.     

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.     

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.     

Biochemical characterization of Sinorhizobium meliloti mutants reveals gene products involved in the biosynthesis of the unusual lipid A very long-chain fatty acid

Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.     

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.     

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.     

BacA-mediated bleomycin sensitivity in Sinorhizobium meliloti is independent of the unusual lipid A modification

Sinorhizobium meliloti bacA mutants are symbiotically defective, deoxycholate sensitive, and bleomycin resistant. We show that the bleomycin resistance phenotype is independent of the lipid A alteration and that the changes giving rise to both phenotypes are likely to be involved in the inability of bacA mutants to persist within their hosts.     

The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.Sinorhizobium meliloti and Brucella abortus form chronic intracellular infections within legumes and mammalian hosts, respectively (3, 20), and their BacA proteins play essential roles in these processes (8, 12). The precise function(s) of the BacA proteins has not been resolved, but free-living S. meliloti and B. abortus mutants lacking BacA have increased resistance to the glycopeptide bleomycin (9, 12) and there are ∼50% decreases in their lipid A very-long-chain fatty acid (VLCFA) contents (4, 7). It has also been determined that the increased resistance of an S. meliloti bacA null mutant to bleomycin and a truncated eukaryotic peptide, Bac7_1-16, is independent of its lipid A VLCFA alteration (6, 15). Together, these findings support a model in which BacA could have multiple nonoverlapping functions which lead to lipid A VLCFA modification and peptide uptake. The fact that two symbiotically defective S. meliloti BacA site-directed mutants (Q193G and R389G) (13) show defects in BacA-mediated lipid A VLCFA modification (4) but are still capable of peptide uptake (15) suggests that the S. meliloti lipid A VLCFA modification could play a key role in the symbiosis of this organism with alfalfa.Since the mechanism by which BacA leads to the lipid A VLCFA modification has not been resolved (4), S. meliloti mutants were constructed with mutations in the lpxXL and acpXL genes, which encode a lipid A VLCFA acyl transferase and a VLCFA acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A (5, 23). The S. meliloti lpxXL and acpXL mutants completely lack the lipid A VLCFA modification in their free-living states, but, unlike the S. meliloti bacA null mutant, these mutants can still form a successful symbiosis with alfalfa (5, 8, 23). However, the fact that the S. meliloti acpXL and lpxXL mutants are substantially less competitive in the alfalfa symbiosis than the parent strain (5, 23) indicates that the AcpXL and LpxXL proteins play important roles in at least one of the stages of the alfalfa symbiosis. Although the free-living S. meliloti acpXL and lpxXL mutants completely lack the lipid A VLCFA, they produce different species of lipid A (5). For example, in the absence of AcpXL, S. meliloti is able to modify lipid A with either C16:0 or C18:0 in the position normally modified with the VLCFA in the parent strain lipid A. This process is LpxXL dependent, as it does not occur in either an S. meliloti lpxXL single mutant or an S. meliloti acpXL lpxXL double mutant. In addition, since a Rhizobium leguminosarum acpXL mutant completely lacks the lipid A VLCFA modification in its free-living state but its lipid A is partially modified with the VLCFA to ∼58% of the amount in the parent strain lipid A during passage through peas (25), it is also possible that the S. meliloti acpXL mutant and possibly the S. meliloti lpxXL mutant undergo further lipid A changes during the interaction with alfalfa.In this study, we found that LpxXL and AcpXL play important but distinct roles in S. meliloti bacteroid development during alfalfa symbiosis. Additionally, we demonstrated that there is a minor host-induced AcpXL-independent mechanism by which S. meliloti bacteroid lipopolysaccharide (LPS) can be modified with the VLCFA. In contrast, we found that the LpxXL protein plays an essential role in the modification of S. meliloti bacteroids with VLCFAs.     

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.     

A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots

Swarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co-ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi-solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild-type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long-chain fatty acyl-CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl-CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.     

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.     

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.     

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.     

Enteric YaiW Is a Surface-Exposed Outer Membrane Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions.