Characterization of hydroxymethylpyrimidine phosphate kinase from mesophilic and thermophilic bacteria and structural insights into their differential thermal stability.

The hydroxymethylpyrimidine phosphate kinases (HMPPK) encoded by the thiD gene are involved in the thiamine biosynthesis pathway, can perform two consecutive phosphorylations of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) and are found in thermophilic and mesophilic bacteria, but only a few characterizations of mesophilic enzymes are available. The presence of another homolog enzyme (pyridoxal kinase) that can only catalyze the first phosphorylation of HMP and encoded by pdxK gene, has hampered a precise annotation in this enzyme family. Here we report the kinetic characterization of two HMPPK with structure available, the mesophilic and thermophilic enzyme from Salmonella typhimurium (StHMPPK) and Thermus thermophilus (TtHMPPK), respectively. Also, given their high structural similarity, we have analyzed the structural determinants of protein thermal stability in these enzymes by molecular dynamics simulation. The results show that pyridoxal kinases (PLK) from gram-positive bacteria (PLK/HMPPK-like enzymes) constitute a phylogenetically separate group from the canonical PLK, but closely related to the HMPPK, so the PLK/HMPPK-like and canonical PLK, both encoded by pdxK genes, are different and must be annotated distinctly. The kinetic characterization of StHMPPK and TtHMPPK, shows that they perform double phosphorylation on HMP, both enzymes are specific for HMP, not using pyridoxal-like molecules as substrates and their kinetic mechanism involves the formation of a ternary complex. Molecular dynamics simulation shows that StHMPPK and TtHMPPK have striking differences in their conformational flexibility, which can be correlated with the hydrophobic packing and electrostatic interaction network given mainly by salt bridge bonds, but interestingly not by the number of hydrogen bond interactions as reported for other thermophilic enzymes. ENZYMES: EC 2.7.1.49, EC 2.7.4.7, EC 2.7.1.35, EC 2.7.1.50.

A feed-forward loop between SroC and MgrR small RNAs modulates the expression of eptB and the susceptibility to polymyxin B in Salmonella Typhimurium.

Base-pairing small RNAs (sRNAs) regulate gene expression commonly by direct interaction with cognate mRNAs. Nevertheless, recent studies have expanded this knowledge with the discovery of the RNA 'sponges' which are able to interact and repress the functions of classical base-pairing sRNAs. In this work, we present evidence indicating that the sponge RNA SroC from Salmonella enterica serovar Typhimurium base pairs with the MgrR sRNA, thereby antagonizing its regulatory effects on both gene expression and resistance to the antimicrobial peptide polymyxin B (PMB). By a predictive algorithm, we determined putative SroC-MgrR base-pairing regions flanking the interaction area between MgrR and its target mRNA, eptB, encoding a LPS-modifying enzyme. With a two-plasmid system and compensatory mutations, we confirmed that SroC directly interacts and down-regulates the levels of MgrR, thus relieving the MgrR-mediated repression of eptB mRNA. Since it was previously shown that an Escherichia coli strain carrying an mgrR deletion is more resistant to PMB, we assessed the significance of SroC in the susceptibility of S. Typhimurium to PMB. Whereas the sroC deletion increased the sensitivity to PMB, as compared to the wild-type, the resistance phenotypes between the ΔmgrR and ΔsroCΔmgrR strains were comparable, evidencing that mgrR mutation is epistatic to the sroC mutation. Together, these results indicate that both SroC and MgrR sRNAs compose a coherent feed-forward loop controlling the eptB expression and hence the LPS modification in S. Typhimurium.

A multi-drug resistant Salmonella Typhimurium ST213 human-invasive strain (33676) containing the bla CMY-2 gene on an IncF plasmid is attenuated for virulence in BALB/c mice.

BACKGROUND: Classical strains of Salmonella enterica serovar Typhimurium (Typhimurium) predominantly cause a self-limiting diarrheal illness in humans and a systemic disease in mice. In this study, we report the characterization of a strain isolated from a blood-culture taken from a 15-year old woman suffering from invasive severe salmonellosis, refractory to conventional therapy with extended-spectrum cephalosporin (ESC). RESULTS: The strain, named 33676, was characterized as multidrug-resistant Salmonella serogroup A by biochemical, antimicrobial and serological tests. Multilocus sequence typing (MLST) and XbaI macrorestrictions (PFGE) showed that strain 33676 belonged to the Typhimurium ST213 genotype, previously described for other Mexican Typhimurium strains. PCR analyses revealed the presence of IncA/C, IncFIIA and ColE1-like plasmids and the absence of the Salmonella virulence plasmid (pSTV). Conjugation assays showed that the ESC-resistance gene bla CMY-2 was carried on the conjugative IncF plasmid, instead of the IncA/C plasmid, as found in previously studied ST213 strains. Although the IncA/C plasmid conferred most of the observed antimicrobial resistances it was not self-conjugative; it was rather able to conjugate by co-integrating with the IncF plasmid. Strain 33676 was fully attenuated for virulence in BALB/c mice infections. Both type-three secretion system (T3SS), encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), were functional in the 33676 strain and, interestingly, this strain produced the H2 FljB flagellin instead of the H1 FliC flagellin commonly expressed by S. enterica strains. CONCLUSIONS: Strain 33676 showed two main features that differentiate it from the originally described ST213 strains: 1) the bla CMY-2 gene was not carried on the IncA/C plasmid, but on a conjugative IncF plasmid, which may open a new route of dissemination for this ESC-resistance gene, and 2) it expresses the H2 FljB flagella, in contrast with the other ST213 and most Typhimurium reference strains. To our knowledge this is the first report of an IncF bla CMY-2-carrying plasmid in Salmonella.

Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

BACKGROUND: Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. RESULTS: YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. CONCLUSIONS: We showed that the transfer of the YU39 blaCMY-2 gene harbored on a non- conjugative pA/C requires the machinery of a highly conjugative pX1 plasmid. Our experiments demonstrate the complex interactions a single strain can exploit to contend with the challenge of horizontal transfer and antibiotic selective pressure.

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a beta-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

The dioxygenase-encoding olsD gene from Burkholderia cenocepacia causes the hydroxylation of the amide-linked fatty acyl moiety of ornithine-containing membrane lipids.

Burkholderia cenocepacia is an important opportunistic pathogen, and one of the most striking features of the Burkholderia genus is the collection of polar lipids present in its membrane, including phosphatidylethanolamine (PE) and ornithine-containing lipids (OLs), as well as the 2-hydroxylated derivatives of PE and OLs (2-OH-PE and 2-OH-OLs, respectively), which differ from the standard versions by virtue of the presence of a hydroxyl group at C2 (2-OH) of an esterified fatty acyl residue. Similarly, a lipid A-esterified myristoyl group from Salmonella typhimurium can have a 2-hydroxy modification that is due to the LpxO enzyme. We thus postulated that 2-hydroxylation of 2-OH-OLs might be catalyzed by a novel dioxygenase homologue of LpxO. In B. cenocepacia, we have now identified two open reading frames (BCAM1214 and BCAM2401) homologous to LpxO from S. typhimurium. The introduction of bcam2401 (designated olsD) into Sinorhizobium meliloti leads to the formation of one new lipid and in B. cenocepacia of two new lipids. Surprisingly, the lipid modifications on OLs due to OlsD occur on the amide-linked fatty acyl chain. This is the first report of a hydroxyl modification of OLs on the amide-linked fatty acyl moiety. Formation of hydroxylated OLs occurs only when the biosynthesis pathway for nonmodified standard OLs is intact. The hydroxyl modification of OLs on the amide-linked fatty acyl moiety occurs only under acid stress conditions. An assay has been developed for the OlsD dioxygenase, and an initial characterization of the enzyme is presented.

The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation.

An initial biochemical characterization of the Salmonella enterica serovar Typhi (S. Typhi) EnvZ sensor protein and several mutant derivatives was performed. Autophosphorylation levels were higher for Escherichia coli EnvZ, intermediate for S. enterica serovar Typhimurium EnvZ and very low for S. Typhi EnvZ, in spite of their high amino acid sequence identity. Consequently, OmpR phosphorylation was related to EnvZ autophosphorylation. Among the mutant derivatives, a C354G mutation in S. Typhi EnvZ resulted in a substantial increase in autophosphorylation, while mutation of its other cysteine residue at position 277 to L or S decreased the EnvZ autophosphorylation level. Upon heterodimerization, the S. Typhi C354G mutant complemented the wild type in vitro, increasing the EnvZ-P yield of both monomers, in accordance with the model where EnvZ autophosphorylation occurs in trans, indicating that dimer formation is a dynamic process. Hence, the C354 and the C277 residues are fundamental in determining the particular intrinsic biochemical characteristics of EnvZ.

Biochemical and structural characterization of Salmonella typhimurium glyoxalase II: new insights into metal ion selectivity.

Glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-beta-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 A shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II.

Replacing the general energy-coupling proteins of the phospho-enol-pyruvate: sugar phosphotransferase system of Salmonella typhimurium with fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars.

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EI(Fructose) and FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria-Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12-14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIA(Glucose), leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.

[Transference of extended-spectrum beta-lactamases from nosocomial strains of Klebsiella pneumoniae to other species of Enterobacteriaceae]. / Transferecia de de beta-lactamasas de espectro extendido desde cepas hospitalarias de Klebsiella pneumoniae a otras especies de enterobacterias.

BACKGROUND: Klebsiella pneumoniae is an important pathogenic bacterium, frequently isolated from nosocomial samples, that exhibits wide antimicrobial resistance profiles, including third generation cephalosporins (3GC), aminoglycosides and quinolones. The resistance to 3GC is mainly due to the synthesis of extended spectrum beta lactamases (ESBL), encoded by conjugative plasmids. AIM: To investigate the potential transference of resistance to 3GC from nosocomial strains of K. pneumoniae to other clinical strains of various species of Enterobacteriaceae. MATERIAL AND METHODS: The mating experiments were carried out in liquid media and three nosocomial strains of K. pneumoniae were used as donors. These strains were ESBL-producers and resistant to, at least, one of the 3GC assayed. One strain of Citrobacter freundii, Salmonella typhimurium, Serratia marcescens and Escherichia coli, isolated from clinical specimens, were used as recipients. The presence of bla genes was investigated by PCR. RESULTS: The three nosocomial strains of K. pneumoniae were able to transfer the resistance to 3GC and the genes encoding the ESBL to the susceptible recipient strains of enterobacteria. The frequency of transference was as high as 3.2 x 10-2 transconjugants/recipient cell when the strain of Citrobacter freundii was used as recipient. All transconjugants exhibited high level of resistance to the 3GC assayed. CONCLUSIONS: Strains of K. pneumoniae isolated from Chilean hospitals are able to disseminate the ESBL genes to clinical strains of others species of Enterobacteriaaceae.

Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity.

N-Nitroso compounds, such as N-nitrosodiethylamine (NDEA), are a versatile group of chemical carcinogens, being suspected to be involved in gastrointestinal tumors in humans. The intestinal microflora can modify a wide range of environmental chemicals either directly or in the course of enterohepatic circulation. Nitroreductases from bacteria seem to have a wide spectrum of substrates, as observed by the reduction of several nitroaromatic compounds, but their capacity to metabolize N-nitroso compounds has not been described. To elucidate the participation of nitroreductase or acetyltransferase enzymes in the mutagenic activity of NDEA, the bacterial (reverse) mutation test was carried out with the strains YG1021 (nitroreductase overexpression), YG1024 (acetyltransferase overexpression), TA98NR (nitroreductase deficient), and TA98DNP6 (acetyltrasferase deficient), and YG1041, which overexpresses both enzymes. The presence of high levels of acetyltransferase may generate toxic compounds that must be eliminated by cellular processes or can lead to cell death, and consequently decrease the mutagenic effect, as can be observed by the comparison of strain TA98DNP6 with the strains TA98 and YG1024. The slope curves for TA98 strain were 0.66 rev/microM (R(2) = 0.51) and 52.8 rev/microM (R(2) = 0.88), in the absence and presence of S9 mix, respectively. For YG1024 strain, the slope curve, in the presence of S9 mix was 6897 rev/microM (R(2) = 0.78). Our data suggest that N-nitroso compounds need to be initially metabolized by enzymes such as cytochromes P450 to induce mutagenicity. Nitroreductase stimulates toxicity, while acetyltransferase stimulates mutagenicity, and nitroreductase can neutralize the mechanism of mutagenicity generating innoccuos compounds, probably by acting on the product generated after NDEA activation.

Transferecia de de ß-lactamasas de espectro extendido desde cepas hospitalarias de Klebsiella pneumoniae a otras especies de enterobacterias / Transference of extended-spectrum ß-lactamases from nosocomial strains of Klebsiella pneumoniae to other species of Enterobacteriaceae

Background: Klebsiella pneumoniae is an important pathogenic bacterium, frequently isolated from nosocomial samples, that exhibits wide antimicrobial resistance profiles, including third generation cephalosporins (3GC), aminoglycosides and quinolones. The resistance to 3GC is mainly due to the synthesis of extended spectrum beta lactamases (ESBL), encoded by conjugative plasmids. Aim: To investigate the potential transference of resistance to 3GC from nosocomial strains of K. pneumoniae to other clinical strains of various species of Enterobacteriaceae. Material and methods: The mating experiments were carried out in liquid media and three nosocomial strains of K. pneumoniae were used as donors. These strains were ESBL-producers and resistant to, at least, one of the 3GC assayed. One strain of Citrobacter freundii, Salmonella typhimurium, Serratia marcescens and Escherichia coli, isolated from clinical specimens, were used as recipients. The presence of bla genes was investigated by PCR. Results: The three nosocomial strains of K. pneumoniae were able to transfer the resistance to 3GC and the genes encoding the ESBL to the susceptible recipient strains of enterobacteria. The frequency of transference was as high as 3.2 x 10-2 transconjugants/recipient cell when the strain of Citrobacter freundii was used as recipient. All transconjugants exhibited high level of resistance to the 3GC assayed. Conclusions: Strains of K. pneumoniae isolated from Chilean hospitals are able to disseminate the ESBL genes to clinical strains of others species of Enterobacteriaaceae.

High-level resistance to mecillinam produced by inactivation of soluble lytic transglycosylase in Salmonella enterica serovar Typhimurium.

By screening for high-level mecillinam resistant derivatives of a low-level resistant strain (cysB403 galE1922 relA21::Tn10) of Salmonella enterica serovar Typhimurium, a MudJ insertion in the gene for soluble lytic transglycosylase (slt) was isolated. This insertion (slt-1::MudJ) increased the resistance to mecillinam of cysB and cysE strains (MIC: about 20-40 microg mL(-1)) to a strikingly high level (MIC: 160 microg mL(-1)). As in Escherichia coli K-12, the slt mutation slightly increased the sensitivity of the wild type and of several strains that carried mutations that did not increase mecillinam resistance. All the strains acquired a spherical cell shape when treated with mecillinam. The effect of slt-1::MudJ was limited to mecillinam, the response to several other antibiotics remaining unaltered by the insertion. The results presented in this paper demonstrate that soluble lytic transglycosylase performs an important role in the response to mecillinam, which only becomes evident when failure of CysB/CysE function causes medium-level resistance. The results also suggest that soluble lytic transglycosylase interacts with, and is partially inhibited by normal lipopolysaccharide.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium.

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host .zntA gene product.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host ·zntA gene product.

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption.

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.

A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium.

A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet. The mutant D220 grows well at 28 degreesC but has a lower growth rate and forms filaments at 37 degreesC. Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced. The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein. The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype. The insertion dam-231 : : Tn10d Tet resulted in a dam "leaky" phenotype since methylated and unmethylated adenines in GATC sequences were present. In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S. typhimurium strain. The wild-type dam gene of S. typhimurium exhibited 82% identity with the Escherichia coli dam gene.

Effect of primary structure modifications in Trypanosoma cruzi neuraminidase/trans-sialidase activities.

Neuraminidases have been implicated in various processes involving the interaction of pathogens and their receptor cells. Trypanosoma cruzi, the agent of Chagas disease, has an unusual neuraminidase, able to transfer bound alpha(2-3)sialic acid to a suitable acceptor rather than to water: the trans-sialidase (TcTS). This enzyme is encoded by a family of several homologous genes, some of them rendering inactive the products. We have shown that enzymatically active proteins have Tyr in position 342, whereas inactive TcTS contain a His342. We have now mutated this Tyr residue to Phe or Thr. Both mutant proteins resulted in enzymatically inactive products. Chimeras expressing parts of Salmonella typhimurium neuraminidase (NANH) and TcTS were constructed. Only the construct containing the complete NANH molecule fused to the last 272 residues of TcTS had neuraminidase activity. However this construct did not present TcTS activity. This finding suggests that other residues present in the homology region are required for TcTS activity.

A new plasmidic cefotaximase from patients infected with Salmonella typhimurium.

Salmonella typhimurium strains resistant to most beta-lactams, co-trimoxazole, tobramycin and gentamicin were isolated from patients in two hospitals in Buenos Aires, Argentina, beginning in August 1990. The patients were suffering from meningitis, septicaemia or enteritis. Therapy including ampicillin, ceftriaxone and gentamicin failed. The strains produced a plasmidic (pMVP-4) extended broad-spectrum beta-lactamase which is more active against cefotaxime than against ceftazidime (Vmax for cefotaxime 350 times higher than for ceftazidime). This cefotaximase demonstrates similarity to the previously described CTX-ase-M-1 from Escherichia coli, but it is distinctly different from CTX-ase-M-1 by its isoelectric point (7.9 for CTX-ase-M-2 in comparison with 8.9 for CTX-ase-M-1) as well as in its lower susceptibility to the beta-lactamase inhibitors sulbactam, clavulanic acid, tazobactam and BRL 42715. Thus, the beta-lactamase produced by S. typhimurium strains from Argentina appears to represent a new member (CTX-ase-M-2) of a novel group of plasmidic extended broad-spectrum beta-lactamases designated as cefotaximases.