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S Maloy

Publications and source records attributed to S Maloy.

At least 19 recordsLinked to original sources
PubMed2001

Substrate recognition by proline permease in Salmonella.

Proline transport is required for catabolism of proline as a carbon, nitrogen, and energy source, and for accumulation of proline during adaptation to osmotic stress. These physiological processes are widespread in nature, and play essential roles in the virulence of both prokaryotic and eukaryotic pathogens. In enteric bacteria, the major proline permease is encoded by the putP gene. To identify the structural features required for substrate recognition by PutP, we assayed the transport and toxicity of a variety of natural and synthetic derivatives of proline. The results indicate that the substrate binding site of proline permease consists of a hydrophobic pocket that accommodates C3, C4, and C5 of the pyrrolidine ring. Both 4- and 5-membered rings fit into the substrate binding pocket, but 6-membered rings are excluded. Analogs with substituents on the C4 position are also excluded. In addition, the binding site includes a hydrophilic region that recognizes the imino and carbonyl groups. A free carboxyl group is not required. Taken together, these results may be used to design new synthetic inhibitors of proline transport that can effectively block proline uptake by microbial pathogens.

Amino Acid Transport Systems, Neutral↗
PubMed2001

Rapid approach to determine rrn arrangement in Salmonella serovars.

A PCR method was developed by which to rapidly and accurately determine the rrn arrangement of Salmonella enterica serovars. Primers were designed to the genomic regions flanking each of the seven rrn operons. PCR analysis using combinations of these primers will distinguish each of the possible arrangements of the rrn skeleton.

Chromosome Mapping↗
PubMed2001

Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences.

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to sef, fim, saf, and tcf, which had been described previously in serotype Typhi, we identified eight new putative chaperone-usher-dependent fimbrial operons, which were termed bcf, sta, stb, ste, std, stc, stg, and sth. Hybridization analysis performed with 16 strains of Salmonella reference collection C and 22 strains of Salmonella reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal Salmonella serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other Salmonella serotypes investigated in that it contained a unique combination of putative fimbrial operons.

Base Sequence↗
PubMed2000

Surrogate genetics: the use of bacterial hybrids as a genetic tool.

Experimental dissection of bacterial genomes requires a well-developed set of genetic tools, but many bacteria lack the essential tools required for genetic analysis. Recombination of a region of chromosomal DNA from poorly characterized donor bacteria with the chromosome of a suitable surrogate host creates a genetically malleable hybrid, providing a short-cut for the detailed genetic analysis of the substituted genes. However, recombination between closely related but nonidentical DNA sequences ("homeologous recombination") is strongly inhibited, posing a powerful barrier to gene exchange between bacteria and a major impediment to the construction of genetic hybrids. By taking advantage of mutS and recD mutant recipients, it is possible to effectively overcome the recombination barrier, allowing construction of genetic hybrids in a related surrogate host. Once stably recombined into the recipient chromosome, the donor DNA can be studied with all the genetic tools available in the surrogate host. In addition to facilitating standard genetic analysis, use of a surrogate host can provide novel approaches to study the physiological roles of unique genes from poorly characterized bacteria.

Bacteria↗
PubMed2000

CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis.

The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild-type B. subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.

Bacillus subtilis↗
PubMed1999

Regulation of flavin dehydrogenase compartmentalization: requirements for PutA-membrane association in Salmonella typhimurium.

PutA is a multifunctional, peripheral membrane protein which functions both as an autogenous transcriptional repressor and the enzyme which catalyzes the two-step conversion of proline to glutamate in Salmonella typhimurium and Escherichia coli. To understand how PutA associates with the membrane, we determined the role of FAD redox and membrane components in PutA-membrane association. Reduction of the tightly bound FAD is required for both derepression of the put operon and membrane association of PutA. FADH(2) alters the conformation of PutA, resulting in an increased hydrophobicity. Previous studies used enzymatic activity as an assay for membrane association and concluded that electron transfer from the reduced FAD in PutA to the membrane is required for the PutA-membrane interaction. However, direct physical assays of PutA association with membrane vesicles from quinone deficient mutants demonstrated that although electron transfer is essential for proline dehydrogenase activity, it is not required for PutA-membrane association per se. Furthermore, PutA efficiently associated with liposomes, indicating that PutA-membrane association does not require interactions with other membrane proteins. PutA enzymatic activity can be efficiently reconstituted with liposomes containing ubiquinone and cytochrome bo, confirming that proline dehydrogenase can pass electrons directly to the quinone pool. These results indicate that PutA-membrane association is due strictly to a protein-lipid interaction initiated by reduction of FAD.

Bacterial Proteins↗
PubMed1999

Effect of mutS and recD mutations on Salmonella virulence.

Hybrid derivatives of closely related bacteria may be used to dissect strain-specific functions that contribute to virulence within a host. However, mismatches between DNA sequences are a potent barrier to recombination. Recipients with mutS and recD mutations overcome this barrier, allowing construction of genetic hybrids. To determine whether Salmonella hybrids constructed in a mutS recD host can be used to study virulence, we assayed the effect of mutS and recD mutations on the virulence of Salmonella typhimurium 14028s in mice. Mutants defective in either mutS or recD do not affect the time course or the 50% lethal dose (LD(50)) of the infection. In contrast, the inactivation of both mutS and recD results in a synthetic phenotype which substantially increases the time required to cause a lethal infection without changing the LD(50). This phenotype results from an inability of mutS recD double mutants to rapidly adapt to purine-limiting conditions present within macrophages. Although the disease progression is slower, S. typhimurium mutS recD mutants retain the ability to cause lethal infections, and, thus, hybrids constructed in mutS recD hosts may permit the analysis of virulence factors in a surrogate animal model.

Adenosine Triphosphatases↗
PubMed1998

The PutA protein of Salmonella typhimurium catalyzes the two steps of proline degradation via a leaky channel.

Proline utilization in Salmonella typhimurium requires two proteins encoded by the put operon: PutP, the major proline permease, and PutA. PutA is a multifunctional, peripheral membrane protein which acts both as a transcriptional repressor for the put operon and enzyme catalyzing the two-step conversion of proline to glutamate. In the first enzymatic reaction catalyzed by PutA, proline oxidation to pyrroline-5-carboxylate (P5C) is coupled with the reduction of a tightly associated FAD. In the second reaction, P5C oxidation to glutamate is coupled with reduction of soluble NAD. Although PutA can use exogenous P5C, the concentration of exogenous P5C required for the P5C dehydrogenase reaction is much greater than the steady-state P5C concentration accumulated during proline degradation. Furthermore, exogenous P5C does not efficiently compete against endogenous P5C for the production of glutamate, and the endogenous P5C produced directly from proline is preferentially used by PutA for the production of glutamate. Kinetic assays indicate that in the presence of NAD the two enzymatic reactions of PutA function synchronously to increase the overall reaction rate over that of the two independent reactions, and the second reaction proceeds in the absence of a lag phase. These results indicate that PutA directly transfers the intermediate P5C between the two enzymatic functions via a "leaky channel" mechanism. Because both the reduction of FAD and the intermediate P5C stimulate membrane association of PutA, channeling of P5C may also contribute to the regulation of proline utilization.

Bacterial Proteins↗
PubMed1998

Genetic analysis, using P22 challenge phage, of the nitrogen activator protein DNA-binding site in the Klebsiella aerogenes put operon.

The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons from Klebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

Amino Acid Transport Systems, Neutral↗
PubMed1997

Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between Salmonella typhimurium and Salmonella typhi.

Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between Escherichia coli and Salmonella typhimurium and between S. typhimurium and Salmonella typhi. However, even in mutants defective for mismatch repair the recombination frequencies are 10(2)- to 10(3)-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between S. typhimurium and S. typhi is less than in transductions between strains of S. typhimurium. In homeologous transductions, mutations in the recD gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in mutS recD double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of mutS and recD act independently. Because S. typhimurium and S. typhi are approximately 98-99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.

Adenosine Triphosphatases↗
PubMed1997

Regulation of gene expression by repressor localization: biochemical evidence that membrane and DNA binding by the PutA protein are mutually exclusive.

The PutA protein from Salmonella typhimurium is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate, a reaction that is coupled to the transfer of electrons to the electron transport chain in the cytoplasmic membrane. The PutA protein is also a transcriptional repressor that regulates the expression of the put operon in response to the availability of proline. Despite extensive genetic and biochemical studies of the PutA protein, it was not known if the PutA protein carries out both of these two opposing functions while membrane associated or if instead it carries them out in different cellular compartments. To distinguish between these alternatives, we directly assayed the binding of purified PutA protein to DNA and membranes in vitro. The results indicate that wild-type PutA does not simultaneously associate with DNA and membranes. In addition, PutA superrepressor mutants that exhibit increased repression of the put genes show a direct correlation between decreased membrane binding and increased DNA binding. These results support a model in which the PutA protein shuttles between the membrane (where it acts as an enzyme but lacks access to DNA-binding sites) and the cytoplasm (where it binds DNA and acts as a transcriptional repressor), depending on the availability of proline.

Bacterial Proteins↗
PubMed1995

Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium.

There exists a plethora of tyrosine kinases that play essential roles in regulation of eukaryotic proteins. Several dual specificity kinases that phosphorylate proteins on threonine, serine, and tyrosine residues also play critical roles in eukaryotic phosphorylation cascades. In contrast, very few prokaryotic proteins have been shown to be phosphorylated on tyrosine residues, and the functions of the rare examples remain obscure. Furthermore, no dual specificity kinases have been described in prokaryotes. Our results indicate that PutA protein from the bacterium Salmonella typhimurium autophosphorylates on several threonine, serine, and tyrosine residues. PutA protein both represses the proline utilization (put) operon and degrades proline to glutamate. These two opposing functions are regulated by the availability of proline and the membrane sites needed for the proline dehydrogenase activity of PutA protein. In addition, these functions are modulated by phosphorylation of PutA protein. The rate of dephosphorylation of PutA protein is determined by the availability of proline and membranes. Dephosphorylated PutA protein has a higher DNA binding affinity than the phosphorylated protein and thus may prevent toxic overexpression of PutA protein in the absence of available membrane sites.

Adenosine Triphosphate↗
PubMed1995

Proline dehydrogenase activity of the transcriptional repressor PutA is required for induction of the put operon by proline.

The proline utilization (put) operon from Salmonella typhimurium consists of the putP gene, encoding a proline transporter, and the putA gene, encoding an enzyme with both proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase activities. In addition to these two enzymatic activities, the PutA protein is a transcriptional repressor that regulates the expression of putP and putA in response to the availability of proline. We report the isolation of super-repressor mutants of PutA that decrease expression from the putA promoter in the presence or absence of proline. None of the mutants exhibited increased affinity for the DNA in the put regulatory region in vitro. Although DNA binding by wild-type PutA was prevented by the addition of proline and an artificial electron acceptor, DNA binding by the two strongest super-repressors was not prevented under identical conditions. The proline dehydrogenase activity of the purified mutant proteins showed altered kinetic properties (increased Km(Pro), reduced Vmax, or a completely null phenotype). The observation that these mutations simultaneously affect induction by proline and proline dehydrogenase activity suggests that a single proline-binding site is involved in both proline dehydrogenase activity and induction of the expression of the put operon. Furthermore, the results indicate that the proline dehydrogenase activity of PutA is essential for induction of the put operon by proline.

Alleles↗
PubMed1995

MudSacI, a transposon with strong selectable and counterselectable markers: use for rapid mapping of chromosomal mutations in Salmonella typhimurium.

The transposable bacteriophage Mu and its mini-Mu derivatives are useful tools for the genetic analysis of many bacteria. A variety of antibiotic-resistant Mu derivatives have been constructed, allowing direct selection for cells which contain the transposon. However, in many cases a counterselection against the transposon would greatly facilitate further genetic analysis. In this paper we report the construction of MudSacI, a mini-Mu derived transposon containing the sacB (secretory levansucrase) gene of Bacillus subtilis, which confers sucrose sensitivity upon gram-negative bacteria. We describe the use of this transposon as a tool for rapid genetic mapping of chromosomal genes in Salmonella typhimurium. Simple modifications of this approach should facilitate rapid mapping in many other bacteria as well.

Bacteriophage mu↗
PubMed1994

Inactivation of mismatch repair overcomes the barrier to transduction between Salmonella typhimurium and Salmonella typhi.

P22 transduction of chromosomal genes from Salmonella typhimurium into Salmonella typhi occurs at a low frequency. Transduction of plasmids from S. typhimurium into S. typhi occurs at a frequency similar to that between S. typhimurium strains, indicating that the barrier to transduction of chromosomal genes is not due to an inability of P22 to inject DNA into S. typhi or a restriction endonuclease that rapidly degrades foreign DNA. Furthermore, transduction of mutS and mutL derivatives of S. typhi with chromosomal genes from S. typhimurium occurs efficiently. These results indicate that the transduction barrier is due to activity of the recipient mismatch repair system, which senses sequence divergence and disrupts heteroduplexes in favor of recipient sequences. Inactivation of the mismatch repair system allows P22 transduction to be used as an effective tool for constructing S. typhi-S. typhimurium hybrids.

DNA Repair↗
PubMed1993

PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator.

The proline utilization (put) operon of Salmonella typhimurium is transcriptionally repressed by PutA protein in the absence of proline. PutA protein also carries out the enzymatic steps in proline catabolism. These two roles require different cellular localizations of PutA. Catabolism of proline requires PutA to associate with the membrane because reoxidation of the FAD cofactor in PutA needs the presence of an electron acceptor. Repression of the put operon requires PutA to bind to the put control-region DNA in the cytoplasm. The presence of proline, the inducer, is necessary but not sufficient for PutA to discriminate between its roles as an enzyme or as a repressor. Two conditions that prevent PutA protein binding to the put control region are (i) when proline and an electron acceptor or the cytoplasmic membrane are present or (ii) when PutA is reduced by dithionite. These two conditions increase the relative hydrophobicity of PutA protein, favoring membrane association and therefore enzymatic activity.

Bacterial Proteins↗