Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons.

The chromosomally encoded galactose utilization (gal) operons of Salmonella typhimurium and S. typhi were each cloned on similar 5.5-kilobase HindIII fragments into pBR322 and were identified by complementation of Gal- Escherichia coli strains. Restriction endonuclease analyses indicated that these Salmonellae operons share considerable homology, but some heterogeneities in restriction sites were observed. Subcloning and exonuclease mapping experiments showed that both operons have the same genetic organization as that established for the E. coli gal operon (i.e., 5' end, promoter, epimerase, transferase, kinase, and 3' end). Two gal operator regions (oE and oI) of S. typhimurium, identified by repressor titration in an E. coli superrepressor [galR(Sup)] mutant, were sequenced and found to flank the promoter region. This promoter region is identical to the -10 and -35 regions of the E. coli gal operon. Minicell studies demonstrated that the three gal structural genes of S. typhimurium encode separate polypeptides of 39 kilodaltons (kDa) (epimerase, 337 amino acids [aa's]), 41 kDa (transferase, 348 aa's), and 43 kDa (kinase, 380 aa's). Despite functional and organizational similarities, DNA sequence analysis revealed that the S. typhimurium gal genes show less than 70% homology to the E. coli gal operon. Because of codon degeneracy, the deduced amino acid sequences of these polypeptides are highly conserved (greater than 90% homology) as compared with those of the E. coli gal enzymes. These studies have defined basic genetic parameters of the gal genes of two medically important Salmonella species, and our findings support the hypothesized divergent evolution of E. coli and Salmonella spp. from a common ancestral parent bacterium.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.     

Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12.

The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18-kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis.     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.     

Genomic Subtraction Identifies Salmonella typhimurium Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon, stf, Which Are Absent in Salmonella typhi

Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.     

Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli.

The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in lambda gtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by lambda Tn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [14C]fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [14C]fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis.     

Facile and gentle method for quantitative lysis of Escherichia coli and Salmonella typhimurium

Garrett et al. (Mol. Gen. Genet. 182:326-331, 1981) constructed strains of Escherichia coli harboring derivatives of plasmid pBR322 that carry the lysis genes (S, R, and Rz) of phage lambda. The plasmid construction placed the genes under control of the lactose operon operator-promotor (and thus of lac repressor). Induction of E. coli strains carrying these plasmids resulted in rapid lysis of the culture unless the S gene was defective, in which case the cells grew normally. A freeze-thaw treatment of induced cells carrying an S- plasmid gave quantitative lysis of either E. coli or Salmonella typhimurium cells under exceptionally gentle conditions. The method was equally effective on exponential phase cells and stationary phase cells and was readily extended to a large number of independent cultures.     

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids.

We have prepared a library of Salmonella typhimurium genomic fragments cloned in pBR322 and packaged in P22HT capsids. Plasmids carrying 24 of 26 specific genes searched for were isolated by transduction at frequencies of 1 to 344 per 10(6) plasmid transductants. All 11 known genes of the cysteine regulon were isolated from this library, including cysK, which we had previously been unable to clone in a recombinant plasmid with an Escherichia coli host. This library provides a simple and rapid method for isolating most S. typhimurium genes by using S. typhimurium itself as a host and should be particularly useful for cloning genes that might be deleterious to E. coli.     

A third family of allelic hsd genes in Salmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA

Salmonella enterica serovar blegdam has a restriction and modification system encoded by genes linked to serB . We have cloned these genes, putative alleles of the hsd locus of Escherichia coli  K-12, and confirmed by the sequence similarities of flanking DNA that the hsd genes of S. enterica serovar blegdam have the same chromosomal location as those of E. coli K-12 and Salmonella enterica serovar typhimurium LT2. There is, however, no obvious similarity in their nucleotide sequences, and while the gene order in S. enterica serovar blegdam is serB hsdM , S and R , that in E. coli K-12 and S. enterica serovar typhimurium LT2 is serB hsdR , M and S . The hsd genes of S. enterica serovar blegdam identify a third family of serB -linked hsd genes (type ID). The polypeptide sequence predicted from the three hsd genes show some similarities (18–50% identity) with the polypeptides of known and putative type I restriction and modification systems; the highest levels of identity are with sequences of Haemophilus influenzae Rd. The HsdM polypeptide has the motifs characteristic of adenine methyltransferases. Comparisons of the HsdR sequence with those for three other families of type I systems and three putative HsdR polypeptides identify two highly conserved regions in addition to the seven proposed DEAD-box motifs.     

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase.

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.     

Electrotransformation in Salmonella typhimurium LT2

Electroporation gives high efficiency of transformation in Salmonella typhimurium LT2, yielding 10(8)-10(9) electrotransformants per microgram of pBR322 DNA. Lipopolysaccharide (LPS) composition has little influence on electrotransformation efficiency by electroporation, unlike Ca2+ shock methods, which give ca. 10(6) transformants/microgram DNA with strains with Rc or Rd2 LPS, 10(4) transformants with most smooth and rough strains, and 10(2) transformants with strains with Re LPS. Thus cell envelope properties are less crucial in electrotransformation than in Ca2+ shock methods. The reciprocal restriction barrier between Escherichia coli K-12 and S. typhimurium LT2 reduces electrotransformation by ca. 100-fold, but host-restriction mutants reduce or eliminate the barrier.     

DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic') plasmid.     

Cloning and DNA sequence analysis of the wild-type and mutant cyclic AMP receptor protein genes from Salmonella typhimurium.

The crp gene from Salmonella typhimurium, as well as two mutant adenylate cyclase regulation genes designated crpacr-3 and crpacr-4, were cloned into the EcoRI site of plasmid pUC8. Initially cloned on 5.6-kilobase fragments isolated from EcoRI digests of chromosomal DNA, these genes were further subcloned into the BamHI-EcoRI site of plasmid pBR322. When tested, Escherichia coli crp deletion strains harboring the clones regained their ability to pleiotropically ferment catabolite-repressible sugars. Also, the crpacr-containing strains displayed sensitivity to exogenous cyclic AMP (cAMP) when grown on eosin-methylene blue medium with xylose as the carbon source. The proteins encoded by the S. typhimurium wild-type and mutant crp genes were found to have similar molecular weights when compared with the wild-type cAMP receptor protein (CRP) from E. coli. DNA sequence analysis of the wild-type crp gene showed only a three-nucleotide difference from the E. coli sequence, suggesting little divergence of the crp gene between these organisms. The crpacr sequences, however, each contained single nucleotide changes resulting in amino acid substitutions at position 130 of the CRP. Based on the site at which these substitutions occur, the crpacr mutations are believed to affect CRP-cAMP interactions.     

Physical map of Salmonella typhimurium LT2 DNA in the vicinity of the proA gene.

More than 55 kilobases of chromosomal DNA of Salmonella typhimurium LT2, including the gpt, proA, ataA, and newD genes, were cloned in plasmid vector pULB113. The locations of the genes and selected restriction endonuclease cleavage sites were established, and some of the restriction enzyme fragments were subcloned in plasmid vector pBR322.