Impact of either elevated or decreased levels of cytochrome bd expression on Shigella flexneri virulence

Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bd is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. The cydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The ribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneri intracellular survival and virulence.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotypically nearly identical, a difference has been reported in the infective dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasiveness of these bacteria toward epithelial cells. Thus, despite the high degree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may exist. We have undertaken a systematic genetic analysis of the EIEC plasmid pSF204, using transposon mutagenesis. Congo red-negative TnphoA insertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were isolated and screened for the ability to invade cultured HEp-2 cells. Most invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mxi, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxiM, and mxiD homologs and in a proposed new gene, named invX, located downstream of the spa region were identified and characterized. This analysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a strict requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.     

Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ- transposable element was constructed to insert the lacZ-(Trp570 --> Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop --> LacZ+; candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14000 colonies, and named plm-1 (preferential lacZ mutagenesis) and plm-2. The frequency of LacZ- --> LacZ+ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac+ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan(r) gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ+ --> LacZ- and Kan(r) --> Kan(s) mutant frequencies of these Lac+ revertants were in the range of 10(-3) to 10(-2), indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

Salmonella typhimurium cob mutants are not hyper-virulent

It was previously reported that Salmonella typhimurium LT2 cob mutants defective in the biosynthesis of vitamin B12 (cobalamin) are more virulent than the wild type in mice. Here we show that the strains used previously are non-isogenic and that the proposed increase in virulence of the cob mutant strain results from an uncharacterized mutation in the "wild type" which attenuates virulence, most likely by decreasing expression of the spv genes on the virulence plasmid. As a result the cob mutant will appear as hyper-virulent. Examination of the virulence of reconstructed wild-type and cob mutant strains showed that their growth rates were similar in mice, and we conclude that vitamin B12 does not affect the virulence of S. typhimurium LT2.     

Inactivation of DsbA, but not DsbC and DsbD, affects the intracellular survival and virulence of Shigella flexneri

In this study, three mutants, dsbA::kan, dsbC-kan, and dsbD-kan, of Shigella flexneri serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Serény test, the dsbA mutant showed reduced virulence while the dsbC and dsbD mutants were similar to the wild type. That inactivation of dsbA was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the dsbA mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, the dsbA mutant was able to produce actin comets and protrusions, indicating its capacity for intra- and intercellular spread. However, a kinetic analysis of intracellular growth showed that the dsbA mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that dsbA mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an icsB mutant reported previously. Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol. Thus, the attenuation mechanism for dsbA mutant appears to be more complicated than was previously suggested.     

Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine

Shigella flexneri 2a strain CVD 1204, which was constructed by introducing a specific, in-frame deletion mutation in the guaB-A operon, was compared with deltaaroA strain CVD 1201. CVD 1204 was less invasive for HeLa cells than CVD 1201, whereas following invasion, the abilities of the two mutants to proliferate intracellularly were similarly impaired. The reduction in invasiveness was independent of the guanine auxotrophic phenotype and fully recovered when the chromosomal deletion mutation in CVD 1204 was repaired. Following inoculation of the conjunctival sac of guinea pigs (Serény test) at high doses (10(9) CFU per eye), both strains evoked minimal, short- lived conjunctival inflammation, which was significantly milder with strain CVD 1204. Double mutant deltaguaB-A deltavirG (also called icsA) strain CVD 1205 induced, after a single intranasal dose, high mucosal immunoglobulin A antilipopolysaccharide titers, which were significantly boosted further following a second dose of vaccine given 14 days later. Upon Serény test challenge with wild-type S. flexneri 2a, CVD 1205-vaccinated animals were significantly protected against keratoconjunctivitis (zero of eight vaccinees versus five of seven controls, P = 0.03; vaccine efficacy, 100%). CVD 1205 is an attractive candidate for human clinical trials.     

Hydrogen sulfide production and fermentative gas production by Salmonella typhimurium require F0F1 ATP synthase activity

A previously isolated mutant of Salmonella typhimurium lacking hydrogen sulfide production from both thiosulfate and sulfite was shown to have a single mutation which also caused the loss of fermentative gas production and the ability to grow on nonfermentable substrates and which mapped in the vicinity of the atp chromosomal locus. The implication that F0F1 ATP synthase might be essential for H2S and fermentative gas production was explored. The phs plasmid conferring H2S production on wild-type Escherichia coli failed to confer this ability on seven of eight E. coli atp point mutants representing, collectively, the eight genes encoding the subunits of F0F1 ATP synthase. However, it did confer some thiosulfate reductase activity on all except the mutant with a lesion in the ATP synthase catalytic subunit. Localized mutagenesis of the Salmonella atp chromosomal region yielded 500 point mutants unable to reduce thiosulfate to H2S or to produce gas from glucose, but differing in the extents of their ability to grow on succinate, to perform proton translocation as measured in a fluorescence quenching assay, and to reduce sulfite to H2S. Biochemical assays showed that all mutants were completely devoid of both methyl viologen and formate-linked thiosulfate reductase and that N,N'-dicyclohexylcarbodiimide blocked thiosulfate reductase activity by the wild type, suggesting that thiosulfate reductase activity has an absolute requirement for F0F1 ATP synthase. Hydrogenase-linked formate dehydrogenase was also affected, but not as severely as thiosulfate reductase. These results imply that in addition to linking oxidation with phosphorylation, F0F1 ATP synthase plays a key role in the proton movement accompanying certain anaerobic reductions and oxidations.     

The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli

In a genetic screen designed to generate Escherichia coli strains completely devoid of the heme precursor 5-aminolevulinic acid (ALA), we isolated a class of mutants which were defective for exogenous ALA uptake. The mutations, designated alu (ALA uptake), mapped to the 80-min region of the E. coli chromosome. They were complemented by a recombinant plasmid containing the dpp operon, which encodes a dipeptide permease transport system. Alu mutants displayed a severe reduction in ALA import, as did a strain with a chromosomal insertion in the first gene of the dpp operon. A recognized substrate of Dpp transport, prolyl-glycine, effectively competed with ALA for uptake. E. coli strains defective in ALA biosynthesis (hemA or hemL) require exogenous ALA to achieve wild-type growth but show limited aerobic and anaerobic growth in the absence of ALA. The presence of an alu or dpp mutation in hemA or hemL strains abolishes growth in the absence of ALA and requires increased levels of ALA for normal growth. We conclude that the alu mutations are within the dpp operon and that the dipeptide transport system mediates uptake of the important metabolite ALA.     

Coupled structure changes of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and delta secG::kan double mutant

An Escherichia coli strain carrying either the secAcsR11 or delta secG::kan mutation is unable to grow at low temperature owing to cold-sensitive protein translocation but grows normally at 37 degree C. However, introduction of the two mutations into the same cells caused a severe defect in protein translocation and the cells were unable to grow at any temperature examined, indicating that secG is essential for the secAcsR11 mutant. The mutant SecA (csSecA) was found to possess a single amino acid substitution in the precursor-binding region and was defective in the interaction with the precursor protein. Furthermore, the membrane insertion of SecA and the membrane topology inversion of SecG, both of which took place upon the initiation of protein translocation, were significantly retarded even at 37 degree C, when csSecA was used instead of the wild-type SecA. The insertion of the wild-type SecA was also significantly defective when SecG-depleted membrane vesicles were used in place of SecG-containing ones. No insertion of csSecA occurred into SecG-depleted membrane vesicles. Examination of in vitro protein translocation at 37 degree C revealed that SecG is essential for csSecA-dependent protein translocation. We conclude that SecG and SecA undergo a coupled structure change, that is critical for efficient protein translocation.     

An epimerase gene essential for capsule synthesis in Vibrio vulnificus

The extracellular capsule polysaccharide (CPS) of Vibrio vulnificus is a primary virulence factor which allows survival of the bacteria in the human host. To study the genes involved in expression of the capsule, we generated mutants that lost the ability to produce CPS following the insertion of a minitransposon into the genome of an encapsulated, clinical strain of V. vulnificus. A genomic region, from one nonencapsulated mutant, containing the transposon and flanking V. vulnificus DNA was cloned, and a probe complementary to the chromosomal DNA immediately adjacent to the transposon was used to locate this fragment in the genome of the encapsulated parent strain. The fragment, which contained a putative capsule gene, was cloned and, when supplied in trans, complemented the mutation in the nonencapsulated mutant to restore capsule production. In addition, virulence studies, using the 50% lethal dose assay, showed that the restoration of capsule production also restored the virulence of the organism. Sequence analysis of the gene disrupted by the transposon revealed that it matched a nucleotide-sugar epimerase of Vibrio cholerae O139, with 75 and 85% identities at the nucleotide and amino acid levels, respectively. In addition, computer analysis recognized epimerases of various organisms as highly similar to the putative epimerase of V. vulnificus. Finally, a combination of PCR amplification and Southern blotting showed that this epimerase is common to at least 10 strains of V. vulnificus that each express a serologically distinct CPS. Our results indicate that the epimerase gene is essential for capsule expression in V. vulnificus.     

Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and of NADH:ubiquinone oxidoreductase (nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365

Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.     

Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy

Salmonella typhimurium exhibits a low-pH-inducible acid tolerance response (ATR) that can protect the adapted cell from severe acid challenge (pH 3.3). It is a two-stage system, with some proteins induced at pH 5.8 (pre-acid shock) and others induced below pH 4.5 (acid shock). The genetics of acid resistance was investigated through the use of a new screening medium. The medium contained 200 microM dinitrophenol (DNP) and was adjusted to pH 4.7 to 4.8. The medium will lower the internal pH of cells to a lethal level. However, cells capable of mounting an ATR will survive longer on this medium than acid-intolerant cells. Using this DNP lethal screening strategy, we isolated several acid-sensitive insertion mutants. Some mutants were defective in the pre-acid shock ATR stage but exhibited a normal or nearly normal post-acid shock-induced acid tolerance (atrB and atrC). Others could not induce acid tolerance by using either pre- or post-acid shock strategies (atrD, atrF, and atrG). The atrB locus was found to be part of a regulon under the control of a trans-acting regulator, atbR. An insertion in atbR caused constitutive acid tolerance because of overexpression of the regulon. Mutations in atrD and atrF affected iron metabolism and, in a manner analogous to ferric uptake regulator (fur) mutations, diminished acid resistance. The atrF mutation mapped within the ent cluster, probably in a fep uptake locus. The atrD locus mapped near metC and may represent an insertion into the S. typhimurium homolog of the Escherichia coli exbB or exbD locus. The mutation in atrC caused extreme UV light sensitivity and proved to occur within the polA (DNA polymerase I) locus. The results support the concept of overlapping acid protection systems in S. typhimurium.     

H-NS regulates DNA repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

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.