Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies.

We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B when expressed from the medium-copy-number plasmid pHSG576. The gene maps to 46 min on the standard genetic map, near the menB gene, and is therefore distinct from the previously described pmrA locus. We have mapped the polymyxin resistance activity to a 1.3-kb ClaI-PvuII fragment which contains a small open reading frame that could encode an 85-amino-acid peptide. When an omega-Tet insertion was made into the putative pmrD open reading frame (pmrD2::omega-Tet), the resulting plasmid no longer conferred polymyxin resistance, whereas an omega-Tet insertion into vector sequences had no effect. Maxicell analysis confirmed that a protein of the expected size is made in vivo. The PmrD protein shows no significant homology to any known protein, but it does show limited homology across the active site of the p15 acid protease from Rous sarcoma virus, indicating that the protein may have proteolytic activity. However, changing the aspartic acid residue at the putative active site to alanine reduced but did not eliminate polymyxin resistance. When pmrD2::omega-Tet replaced the chromosomal copy of pmrD, the resulting strain showed wild-type sensitivity to polymyxin and could be complemented to resistance by a plasmid that carried pmrD. The pmrA505 allele confers resistance to polymyxin when present in single copy on the chromosome or when present on a plasmid in pmrA+ pmrD+ cells. In combination with the pmrD(2)::-Tet mutation, the effect o the pmrA505 allele on polymyxin resistance was reduced, whether pmrA505 was present in the chromosome or on a plasmid. Conversely, a strain carrying an insertion in pmrA could be complemented to polymyxin resistance by a plasmid carrying the pmrA505 allele but not by a plasmid carrying pmrD. On the basis of these results, we suggest that polymyxin resistance is mediated by an interaction between PmrA or a PmrA-regulated gene product and PmrD.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.     

Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance.

Overexpression of major outer membrane protein OprH of Pseudomonas aeruginosa as a result of mutation (in strain H181) or adaptation to low Mg2+ concentrations (in parent strain H103) is accompanied by increased resistance to polymyxin B, gentamicin, and EDTA. A 2.8-kb EcoRI fragment containing the oprH gene was subcloned into several different expression plasmids in Escherichia coli. These experiments showed that significant levels of OprH could be produced from a promoter on the EcoRI fragment; that the cloned oprH gene was not regulated by Mg2+ deficiency; that there were no differences in the expression of OprH in any construction, regardless of whether the gene from strain H103 or its OprH-overexpressing, polymyxin B-resistant derivative, strain H181, was used; and that overexpression of OprH in E. coli to the level observed in P. aeruginosa H181 did not result in a resistance phenotype. These results favored the conclusion that the mutation in strain H181 was a regulatory rather than a promoter mutation. The oprH gene was cloned behind the benzoate-inducible pm promoter in plasmid pGB25 and transferred to P. aeruginosa H103. Overexpression of OprH from the cloned gene in H103/pGB25 resulted in EDTA resistance but not polymyxin B resistance. This result suggested that another factor, possibly lipopolysaccharide, was affected by the mutation in strain H181. Consistent with this suggestion was the demonstration that mutants of strain H181 with alterations in lipopolysaccharide had reverted to wild-type polymyxin B susceptibility but had unaltered gentamicin and EDTA resistance. These data were consistent with the hypothesis that OprH replaces outer membrane-stabilizing divalent cations.     

Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrA(C) strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3'-acyloxyacyl-linked myristate (C(14)) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group.     

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica

The PmrA/PmrB regulatory system of Salmonella enterica controls the modification of lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown. Here we address both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin B resistance. The PmrC protein consists of an N-terminal region with five transmembrane domains followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for PmrA-regulated polymyxin B resistance.     

A pmrA constitutive mutant sensitizes Escherichia coli to deoxycholic acid

An Escherichia coli mutant was isolated and shown to be polymyxin B resistant. Mapping and sequence analysis revealed a missense mutation at codon 53 within the pmrA (basR) gene that results in a G-to-V substitution. Fusions of promoters from the pmrC, yibD, and pmrH genes with the lacZ reporter showed that they were constitutively expressed in pmrA53 cells. In pmrA+ strains, these promoters were induced by iron and zinc, while a DeltapmrA mutation blocked induction. The PmrA regulon regulates genes whose products remodel the composition and charge of lipid A and hence the barrier properties of the outer membrane. Along these lines, the pmrA53 mutant was also found to be hypersensitive to the anionic bile detergent deoxycholic acid.     

Characterisation of a repressor gene (xre) and a temperature-sensitive allele from the Bacillus subtilis prophage, PBSX

The defective prophage of Bacillus subtilis 168, PBSX, is a chromosomally based element which encodes a non-infectious phage-like particle with bactericidal activity. PBSX is induced by agents which elicit the SOS response. In a PBSX thermoinducible strain which carries the xhi1479 mutation, PBSX is induced by raising the growth temperature from 37 degrees C to 48 degrees C. A 1.2-kb fragment has been cloned which complements the xhi1479 mutation. The nucleotide sequence of this fragment contains an open reading frame (ORF) which encodes a protein of 113 amino acids (aa). This aa sequence resembles that of other bacteriophage repressors and suggests that the N-terminal region forms a helix-turn-helix motif, typical of the DNA-binding domain of many bacterial regulatory proteins. The ORF is preceded by four 15-bp direct repeats, each of which contains an internal palindromic sequence, and by sequences resembling a SigA-dependent promoter. The nt sequence of an equivalent fragment from the PBSX thermoinducible strain has also been determined. There are three aa differences within the ORF compared to the wild type, one of which lies within the helix-turn-helix segment. This ORF encodes a repressor protein of PBSX.     

Release of the lipopolysaccharide deacylase PagL from latency compensates for a lack of lipopolysaccharide aminoarabinose modification-dependent resistance to the antimicrobial peptide polymyxin B in Salmonella enterica

Salmonella enterica modifies its lipopolysaccharide (LPS), including the lipid A portion, to adapt to its environments. The lipid A 3-O-deacylase PagL exhibits latency; deacylation of lipid A is not usually observed in vivo despite the expression of PagL, which is under the control of a two-component regulatory system, PhoP-PhoQ. In contrast, PagL is released from latency in pmrA and pmrE mutants, both of which are deficient in aminoarabinose-modified lipid A, although the biological significance of this is not clear. The attachment of aminoarabinose to lipid A decreases the net anionic charge at the membrane's surface and reduces electrostatic repulsion between neighboring LPS molecules, leading to increases in bacterial resistance to cationic antimicrobial peptides, including polymyxin B. Here we examined the effects of the release of PagL from latency on resistance to polymyxin B. The pmrA pagL and pmrE pagL double mutants were more susceptible to polymyxin B than were the parental pmrA and pmrE mutants, respectively. Furthermore, introduction of the PagL expression plasmid into the pmrA pagL double mutant increased the resistance to polymyxin B. In addition, PagL-dependent deacylation of lipid A was observed in a mutant in which lipid A could not be modified with phosphoethanolamine, which partly contributes to the PmrA-dependent resistance to polymyxin B. These results, taken together, suggest that the release of PagL from latency compensates for the loss of resistance to polymyxin B that is due to a lack of other modifications to LPS.     

The PmrA-PmrB two-component system responding to acidic pH and iron controls virulence in the plant pathogen Erwinia carotovora ssp. carotovora

Efficient response to environmental cues is crucial to successful infection by plant-pathogenic bacteria such as Erwinia carotovora ssp. carotovora. The expression of the main virulence genes of this pathogen, encoding extracellular enzymes that degrade the plant-cell wall, is subject to complex regulatory machinery where two-component systems play an important role. In this paper, we describe for the first time the involvement of the PmrA-PmrB two-component system in regulation of virulence in a plant-pathogenic bacterium. Disruption of pmrB resulted in reduced virulence both in potato and in Arabidopsis. This is apparently due to reduced production of the extracellular enzymes. In contrast, a pmrA mutant exhibited increased levels of these enzymes implying negative regulation of the corresponding genes by PmrA. Furthermore, the pmrB but not pmrA mutant exhibited highly increased resistance to the cationic antimicrobial peptide polymyxin B suggesting alterations in cell surface properties of the mutant. A similar increase of polymyxin resistance was detected in the wild type at mildly acidic pH with low Mg2+. Functional pmrA is essential for bacterial survival on excess iron at acidic pH, regardless of the Mg2+ concentration. We propose that PmrA-PmrB TCS is involved in controlling of bacterial response to external pH and iron and is crucial for bacterial virulence and survival in planta.     

The mutK Gene of Vibrio cholerae: a New Gene Involved in DNA Mismatch Repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Salmonella recD mutations increase recombination in a short sequence transduction assay.

We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.     

Nucleotide sequence of the gene and features of the major outer membrane protein of a virulent Rickettsia prowazekii strain.

We have determined the nucleotide sequence of the gene for a major outer membrane protein (MOMP) of apparent molecular weight 29.5 kD of the virulent Breinl strain of Rickettsia prowazekii. The gene contains an open reading frame (ORF) that encodes a 282-amino-acid polypeptide with a calculated molecular mass of 31549 daltons. A signal-like peptide sequence is found at the deduced N terminus. A heterologous 29.5-kD antigen expressed in Escherichia coli was shown to be secreted into the periplasm. A database search for similar protein sequences revealed considerable homology of the polypeptide with the E. coli peptidyl-prolyl cis/trans isomerase and related proteins of the parvulin family. The genes for MOMP of the virulent Breinl and EVir strains and the vaccine Madrid E strain were amplified using specific primers and cloned into expression vector pQE-30. We found that the polypeptides encoded by the recombinant DNAs do not differ in SDS-PAGE mobility, while the native MOMP of the Breinl strain is known to be different from the corresponding proteins of the Madrid E and EVir strains. Furthermore, no differences within the ORF for the 29.5-kD proteins of the three strains were found by restriction endonuclease analysis of polymerase chain reaction (PCR) products. A possible role of parvulin-like protein (Plp) in the virulence of epidemic typhus agent and the nature of interstrain differences are discussed. Near the plp gene on the opposite strand, an origin of the gene that codes for the SecA subunit of a preprotein translocase was found.     

The avirulence gene avrBs1 from Xanthomonas campestris pv. vesicatoria encodes a 50-kD protein

A gene cloned from Xanthomonas campestris pv. vesicatoria race 2, avrBs1, specified avirulence on pepper cultivars containing the resistance gene Bs1. A series of exonuclease III deletions were made on a 3.2-kbp DNA fragment that determined full avirulence activity, observed as hypersensitive response (HR) induction. The deletion products were subcloned into the broad host range cloning vector pLAFR3, conjugated into a virulent X. c. pv. vesicatoria race 1 strain, 82-8, and scored for their ability to induce a HR on a pepper cultivar (ECW10R) containing the resistance gene Bs1. A span of approximately 1.8 kbp of DNA was necessary for full induction of the HR. The nucleotide sequence revealed two open reading frames (ORFs) capable of encoding proteins of 12.3 and 49.8 kD, designated ORF1 and ORF2, respectively. Deletions into ORF1 altered the HR-inducing activity to give an intermediate phenotype. Deletions into ORF2 completely destroyed activity. When the ORF2 coding region was driven by the lacZ promoter on plasmid pLAFR3 (placD), full avirulence activity was restored, indicating that ORF2 alone can induce the HR. Antisera raised to a beta-galactosidase-ORF2 fusion protein reacted with a 50-kD protein in X. c. pv. vesicatoria race 1 (placD) transconjugants. The deduced amino acid sequence of ORF2 had approximately 47% overall homology to the carboxyl terminus of the avirulence gene, avrA, isolated from Pseudomonas syringae pv. glycinea race 6, and 86% homology over a region of 49 amino acids. P. s. pv. glycinea, however, did not induce an HR on ECW10R plants.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli.

Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.     

The swi4+ gene of Schizosaccharomyces pombe encodes a homologue of mismatch repair enzymes.

The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching.     

鸭副粘病毒WF01 D株F基因的测序与蛋白特性分析

用鸭副粘病毒凤阳分离株WF01 D作9～10日龄SPF鸡胚的尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF01 D病毒的F基因,获得了1条长约1．7kb的特异性条带。用PCR产物直接测序。测序结果表明,扩增片段大小为1782bp,含有1个1662bp的开放性阅读框,编码554个氨基酸。核苷酸同源性分析表明：WF01 D株与国内外其他NDVF基因的同源性为84．5％～97．0％,其中与国内标准强毒株F48E9的同源性为86．6％,说明WF01 D与国内外的传统毒株有较大变异。与Taiwan95株和J株的同源性为93．6％和97．0％,说明WF01 D与Taiwan95株和J株亲缘关系较近,具有较高的相似性。F蛋白裂解位点的氨基酸顺序为112Arg-Arg-Gln-Lys-Arg-Phe117,表明为NDV的强毒株。蛋白疏水性和抗原性分析表明与标准强毒F48E9株相比没有太大的变异。