Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli.

The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.     

Escherichia coli transport mutants lacking binding protein and other components of the branched-chain amino acid transport systems.

Further evidence on the role of binding proteins in branched-chain amino acid transport in Escherichia coli was obtained by selecting mutants with altered expression of the binding proteins. The mutator phage Mu was used to induce E. coli L-valine-resistant mutants defective in branched-chain amino acid transport. By making use of mild selective conditions and strain backgrounds with derepressed high-affinity, binding protein-mediated transport systems, we were able to isolate a new class of transport mutants defective in these systems. Mutant strains AE84084 (livK::Mucts) and AE840102 (livJ) were found to be defective in leucine-specific and LIV binding proteins, respectively, by transport assay, in vitro binding activity, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mutant strain AE4107 (livH::Mu), although lacking high-affinity, branched-chain amino acid transport, retained functional binding proteins and therefore evidently codes for an additional component of high-affinity transport. The livH, livJ, and livK mutations were mapped by transduction and shown to be closely linked to each other in the malT region (min 74) of the E. coli genetic map.     

Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.     

Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system.

A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated. The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC. The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG. The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively. The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems. The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites. By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively. These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue.     

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.     

Characterization of trbC, a new F plasmid tra operon gene that is essential to conjugative transfer.

We have characterized a previously unidentified gene, trbC, which is contained in the transfer region of the Escherichia coli K-12 fertility factor, F. Our data show that the trbC gene is located between the F plasmid genes traU and traN. The product of trbC was identified as a polypeptide with an apparent molecular weight (Ma) of 23,500 that is processed to an Ma-21,500 mature protein. When ethanol was present, the Ma-23,500 polypeptide accumulated; the removal of ethanol resulted in the appearance of the processed mature protein. Subcellular fractionation experiments demonstrated that the processed, Ma-21,500 mature protein was located in the periplasm. DNA sequence analysis showed that trbC encodes a 212-amino-acid Mr-23,432 polypeptide that could be processed to a 191-amino-acid Mr-21,225 mature protein through the removal of a typical amino-terminal signal sequence. We also constructed two different Kmr gene insertion mutations in trbC and crossed these onto the transmissible F plasmid derivative pOX38. We found that cells carrying pOX38 trbC mutant plasmids were transfer deficient and resistant to infection by F-pilus-specific phages. Transfer proficiency and bacteriophage sensitivity were restored by complementation when a trbC+ plasmid clone was introduced into these cells. These results showed that trbC function is essential to the F plasmid conjugative transfer system and suggested that the TrbC protein participates in F-pilus assembly.     

Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.     

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.     

The nucleotide sequence of a small cryptic plasmid found in Staphylococcus aureus and its relationship to other plasmids

The nucleotide sequence of a small (1273 bp) plasmid (pOX1000) of Staphylococcus aureus has been determined and compared with similar plasmids. The sequence includes a single open reading frame; two large palindromes and a 22 bp palindrome that is contiguously repeated three times upstream of the open reading frame. Composite plasmids of pE194ts and pOX1000 were constructed with pUC18 separately inserted into five different sites on pOX1000 and used to analyse the replication functions of the cryptic plasmid.     

Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli.

Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends.     

Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport

Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity of C. jejuni 81-176 in chicks. Cjj81176_1038 is the first gene of a six-gene locus that encodes homologous components of the E. coli LIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system in C. jejuni responsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable for in vivo growth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant for C. jejuni during commensalism.     

Genetic analysis of the Pseudomonas aeruginosa PAO high-affinity branched-chain amino acid transport system by use of plasmids carrying the bra genes.

About 30 mutants of Pseudomonas aeruginosa PAO defective in the high-affinity branched-chain amino acid transport system (LIV-I) were isolated by the selection for resistance to 4-aza-DL-leucine, a toxic leucine analog for LIV-I. All of the mutants were complemented by plasmid pKTH24, harboring the braC gene, which encodes the branched-chain amino acid-binding protein, and the four open reading frames named braD, braE, braF, and braG (T. Hoshino and K. Kose, J. Bacteriol. 172:5531-5539, 1990). We identified five cistrons corresponding to these bra genes by complementation analysis with various derivatives of pKTH24, confirming that the braD, braE, braF, and braG genes are required for the LIV-I transport system. We also found mutations that seem likely to be mutations in a promoter region for the bra genes and those with polarity in the intercistronic region between braC and braD. Analysis with an omega interposon showed that the bra genes are organized as an operon and are cotranscribed in the order braC-braD-braE-braF-braG from a promoter located in the 5'-flanking region of the braC gene.     

Complementation analysis of derepressed mutants of F-like plasmids of Escherichia coli

Complementation analysis of a number of conjugative transfer functions was performed in derepressed (drd) mutants of E. coli F-like plasmids. The major part of double plasmid complexes investigated has revealed the formation of complementation transfer inhibitor of Fin V-type, or less frequently--the formation of Fin U-type inhibitor. An additional complementation analysis of drd plasmids defective at Fin V region genes has demonstrated at least three genes (denoted A, B, C) in the structure of this region.     

Sequence alterations affecting F plasmid transfer gene expression: a conjugation system dependent on transcription by the RNA polymerase of phage T7

We constructed derivatives of the Escherichia coli conjugative plasmid F that carry altered sequences in place of the major transfer operon promoter, PY. Replacement of PY with a promoter-deficient sequence resulted in a transfer-deficient, F-pilus-specific phage-resistant plasmid (pOX38-tra701) that could still express TraJ and TraT; TraY, F-pilin, TraD, and TraI were not detectable on Western blots. On a second plasmid (pOX38-tra715) we replaced PY with a phage T7 late promoter sequence. In hosts carrying a lacUV5-promoter-regulated T7 RNA polymerase gene, all transfer-associated properties of pOX38-tra715 could be regulated with IPTG. After induction, pOX38-tra715 transferred at the wild-type frequency, expressed normal numbers of F-pili and conferred sensitivity to pilus-specific phages. No adverse effects on cell viability were apparent, and additional mutations could easily be crossed onto pOX38-tra715. A traJ deletion (pOX38-tra716) had no effect on the IPTG-induced transfer phenotype. Insertion of cam into trbC, resulted in a mutant (pOX38-tra715trbC33) which, after induction, exhibited the same phenotype associated with other trbC mutants; it could also be complemented by expression of trbC in trans. With pOX38-tra715 or its derivatives, we were able to label specifically the products of tra genes located throughout the long tra operon, by using rifampicin. This feature can be used to investigate transfer protein interactions and to follow changes in these proteins that are associated with conjugal mating events.