bluuebunny/crossref_metadata_2025 huggingface dataset:
The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria
Authors: Muro-Pastor M I , Reyes J C , Florencio F J
Published: November 2016

NADP+-isocitrate dehydrogenase (NADP+-IDH) activity and protein levels in crude extracts from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and the filamentous, dinitrogen-fixing Anabaena sp. strain PCC 7120 were determined under different nitrogen conditions. The highest NADP+-IDH activity and protein accumulation were found under dinitrogen-fixing conditions for the Anabaena strain and under nitrogen starvation for Synechocystis sp. PCC 6803. The icd gene that encodes the NADP+-IDH from Synechocystis sp. strain PCC 6803 was cloned by heterologous hybridization with the previously isolated icd gene from Anabaena sp. strain PCC 7120. The two cyanobacterial icd genes show 81% sequence identity and share a typical 44-amino-acid region different from all the other icd genes sequenced so far. The icd gene seems to be essential for Synechocystis growth since attempts to generate a completely segregated icd mutant were unsuccessful. Transcripts of 2.0 and 1.6 kb were detected by Northern (RNA) blot analysis, for the Anabaena and Synecho-cystis icd genes, respectively. Maximal icd mRNA accumulation was reached after 5 It of nitrogen starvation in Synechocystis cells and under dinitrogen-fixing conditions in Anabaena cells. Primer extension analysis showed that the structure of the Synechocystis icd gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified Synechocystis NtcA protein binds to the promoter of the icd gene. All these data suggest that the expression of the icd gene from Synechocystis sp. strain PCC 6803 may be subjected to nitrogen control mediated by the positively acting regulatory protein NtcA.
Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants
Authors: Clerget M , Boccard F
Published: November 2016

Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth. The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q. This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif. We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site. By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene. We have isolated three independent phage mutants that are able to grow on an IHF- host. They carry different mutations scattered in the roi gene and specifying single amino-acid changes. The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type. Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA. The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed.
A NusG-like protein from Thermotoga maritima binds to DNA and RNA
Authors: Liao D , Lurz R , Dobrinski B , Dennis P P
Published: November 2016

The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.
Sodium-coupled energy transduction in the newly isolated thermoalkaliphilic strain LBS3
Authors: Prowe S G , van de Vossenberg J L , Driessen A J , Antranikian G , Konings W N
Published: November 2016

Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50 degrees C. Since a high concentration of Na+ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na+ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na+ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na+ and Li+ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed.
Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains
Authors: Zhang X , Lu Q , Inouye M , Mathews C K
Published: November 2016

Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.
Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli
Authors: Rajkumari K , Kusano S , Ishihama A , Mizuno T , Gowrishankar J
Published: November 2016

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the sigma(s) and sigma70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of sigma(s) selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.
Isolation and analysis of a fur mutant of Neisseria gonorrhoeae
Authors: Thomas C E , Sparling P F
Published: November 2016

The pathogenic Neisseria spp. produce a number of iron-regulated gene products that are thought to be important in virulence. Iron-responsive regulation of these gene products has been attributed to the presence in Neisseria spp. of the Fur (ferric uptake regulation) protein. Evidence for the role of Fur in neisserial iron regulation has been indirect because of the inability to make fur null mutations. To circumvent this problem, we used manganese selection to isolate missense mutations of Neisseria gonorrhoeae fur. We show that a mutation in gonococcal fur resulted in reduced modulation of expression of four well-studied iron-repressed genes and affected the iron regulation of a broad range of other genes as judged by two-dimensional polyacrylamide gel electrophoresis (PAGE). All 15 of the iron-repressed spots observed by two-dimensional PAGE were at least partially derepressed in the fur mutant, and 17 of the 45 iron-induced spots were affected by the fur mutation. Thus, Fur plays a central role in regulation of iron-repressed gonococcal genes and appears to be involved in regulation of many iron-induced genes. The size and complexity of the iron regulons in N. gonorrhoeae are much greater than previously recognized.
Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog
Authors: Yuan Y , Crane D D , Barry C E
Published: November 2016

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.
Isolation and characterization of the VnfEN genes of the cyanobacterium Anabaena variabilis
Authors: Thiel T
Published: November 2016

The filamentous cyanobacterium Anabaena variabilis fixes nitrogen in the presence of vanadium (V) and in the absence of molybdenum (Mo), using a V-dependent nitrogenase (V-nitrogenase) encoded by the vnfDGK genes. Downstream from these genes are two genes that are similar to the vnfEN genes of Azotobacter vinelandii. Like the vnfDGK genes, the vnfEN genes were transcribed in the absence of Mo, whether or not V was present. A mutant with an insertion in the vnfN gene lacked V-nitrogenase activity; thus, the vnfEN genes were essential for the V-nitrogenase system in A. variabilis. Growth and acetylene reduction assays with wild-type and mutant strains suggested that the V-nitrogenase reduced dinitrogen better than acetylene. The similarity of the vnfEN genes of A. variabilis and A. vinelandii was not strong. The vnfEN genes of A. variabilis showed greater similarity to the vnfDK genes just upstream than to the A. vinelandii vnfEN genes. Sequence comparisons provide support for the idea that if the vnf genes were transferred laterally among bacterial strains, the vnf cluster was not transferred intact. It appears likely that the structural genes were transferred before a duplication event led to the evolution of the vnfEN genes independently in the two strains. The divergence of the vnfEN genes from the vnfDK genes suggests that this duplication, and hence the transfer of vnf genes, was an ancient event.
Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae
Authors: Levin J C , Stein D C
Published: November 2016

Neisseria gonorrhoeae WS1 is a spontaneous pyocin (a bacteriocin produced by Pseudomonas aeruginosa)-resistant mutant of N. gonorrhoeae FA19 that produces a truncated lipooligosaccharide (LOS) and is non-transformable. The LOS-specific mutation in WS1 was moved into a transformable background by transforming FA19 with chromosomal DNA from WS1 (generating strain JWS-1). A clone (pJCL2) capable of restoring JWS-1 to wild-type LOS expression, as detected by its acquisition of reactivity with monoclonal antibodies and by its complemented sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, was isolated. Sequential unidirectional deletion and DNA sequence analysis of pJCL2 identified an open reading frame, designated lsi-7, that could complement the defect in JWS-1. Homology searches against various databases indicated that lsi-7 bad homology with several Escherichia coli genes involved in the phosphorylation of sugars. lsi-7 is adjacent to the lsi-6 gene, another gene involved in LOS biosynthesis. Complementation studies using Salmonella typhimurium lipopolysaccharide mutants showed lsi-6 and lsi-7 to be gonococcal homologs of S. typhimurium rfaD and rfaE, respectively. Reverse transcriptase PCR analysis demonstrated that lsi-6 and lsi-7 are part of the same transcriptional unit.
Effect of nitrate on the autotrophic metabolism of the acetogens Clostridium thermoautotrophicum and Clostridium thermoaceticum
Authors: Fröstl J M , Seifritz C , Drake H L
Published: November 2016

Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H2-CO2-dependent growth. Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources. This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H2-CO2 did not replace CO. These results indicated that nitrate blocked the reduction of CO2 to the methyl and carbonyl levels. On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive. Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway. However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy. Collectively, these findings indicated that (i) C. thermoautotrophicum and C. thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.
Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum
Authors: Limberger R J , Slivienski L L , El-Afandi M C , Dantuono L A
Published: November 2016

A locus encoding polypeptides associated with flagellar structure and function was identified, sequenced, and characterized in Treponema phagedenis and Treponema pallidum. This locus includes homologs of the FlgD, FlgE, MotA, MOB, FliL, and FliM polypeptides found in Salmonella typhimurium and Bacillus subtilis. These polypeptides are extensively conserved between the two treponemes. Several additional polypeptides or unknown function, including Tapl, located upstream of FlgD, and ORF4, located between FlgE and MotA, were also identified. Transcription analysis using RNA PCR indicated that these genes are likely transcribed as part of a single operon and comprise the 5' region of the treponemal fla operon. Primer extension analysis identified a putative promoter, preceding T. phagedenis tap1 in a region of divergent transcription. Pfla resembles the class II or class III motility-related promoters of S. typhimurium. FlgE and Tap1 were further characterized. Western blotting (immunoblotting) indicated that T. pallidum FlgE exhibited an unusual polypeptide ladder that was similar but not identical to that of T. phagedenis. Triton X-114 phase partitioning of T. phagedenis cells coupled with Western blotting revealed that Tap1 was located in the aqueous phase. Computer analysis indicated that Tap1 had no significant membrane spanning regions, suggesting that it resides primarily in the cytoplasm. The organization and expression of this operon are similar in both treponemes but different from those of previously described motility-related operons. These results indicate that despite extensive amino acid sequence conservation, the expression of spirochete flagellar polypeptides is different from that in other bacteria.
Insertional inactivation of Streptococcus pyogenes sod suggests that prtF is regulated in response to a superoxide signal
Authors: Gibson C M , Caparon M G
Published: November 2016

In establishing an infection, Streptococcus pyogenes has the capacity to bind to the host extracellular matrix protein fibronectin via its protein F adhesin. Previous studies have suggested that the expression of protein F is stimulated during aerobic growth or upon addition of superoxide-generating agents to the culture under O2-limited conditions. To further explore the role of superoxide, we have examined the transcription of the gene which encodes protein F (prtF), as well as the expression of superoxide dismutase (SOD) under conditions which promote or repress protein F expression. These studies show that prtF transcription is regulated in response to superoxide concentration and that SOD is regulated in different environments in a manner which directly parallels the expression of protein F. A mutant deficient in SOD activity was constructed by insertional mutation into the gene which encodes SOD (sod). The resulting mutant was sensitive to superoxide and aerobic conditions, showed hypersensitive induction of prtF in response to superoxide, and expressed prtF under normally unfavorable O2-limited conditions. These findings suggest that a streptococcal signal transduction system which senses superoxide may coordinately control expression of prtF and sod.
The genes for phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii subsp. bulgaricus constitute an operon
Authors: Branny P , De La Torre F , Garel J R
Published: November 2016

In Lactobacillus delbrueckii subsp. bulgaricus, the pyk gene coding for pyruvate kinase and the pfk gene coding for phosphofructokinase formed a bicistronic operon transcribed into a 2.9-kb RNA. The nucleotide sequence of the pyk gene indicated that the encoded protein possessed an extra C-terminal domain with a potential phosphoenolpyruvate-dependent autophosphorylation site.
Glutathione amide and its perthiol in anaerobic sulfur bacteria
Authors: Bartsch R G , Newton G L , Sherrill C , Fahey R C
Published: November 2016

Chromatium species produced the novel biological thiol glutathione amide, gamma-L-glutamyl-L-cysteinylglycine amide (GASH), when grown photoheterotrophically. GASH was largely converted to the corresponding perthiol during photoautotrophic growth on sulfide, suggesting that GASH may have a function in anaerobic sulfide metabolism. This unprecedented form of glutathione metabolism was probably present in anaerobic ancestors of modern cyanobacteria and purple bacteria.
The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage
Authors: Navas M A , Gancedo J M
Published: November 2016

The question of how the loss of regulatory mechanisms for a metabolic enzyme would affect the fitness of the corresponding organism has been addressed. For this, the fructose-1,6-bisphosphatase (FbPase) from Saccharomyces cerevisiae has been taken as a model. Yeast strains in which different controls on FbPase (catabolite repression and inactivation; inhibition by fructose-2,6-bisphosphate and AMP) have been removed have been constructed. These strains express during growth on glucose either the native yeast FbPase, the Escherichia coli FbPase which is insensitive to inhibition by fructose-2,6-bisphosphate, or a mutated E. coli FbPase with low sensitivity to AMP. Expression of the heterologous FbPases increases the fermentation rate of the yeast and its generation time, while it decreases its growth yield. In the strain containing high levels of an unregulated bacterial FbPase, cycling between fructose-6-phosphate and fructose-1,6-bisphosphate reaches 14%. It is shown that the regulatory mechanisms of FbPase provide a slight but definite competitive advantage during growth in mixed cultures.
A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains
Authors: Franco A V , Liu D , Reeves P R
Published: November 2016

The modal distribution of O-antigen chain length is determined by the Wzz (Cld/Rol) protein in those cases in which it has been studied. The system of O-antigen synthesis in Escherichia coli serotypes O8 and O9 is different from that reported for most other bacteria, and chain length distribution is thought not to be determined by a Wzz protein. We report the existence in E. coli O8 and O9 strains of wzz genes which are very similar to and have sequences within the range of variation of those which determine the chain length of typical O antigens. We also find that wzz genes previously identified by their effect on O-antigen chain length, when cloned and transferred to O8 and O9 strains, affect the chain length of a capsule-related form of LPS, K(LPS). We conclude that in at least some O8 and O9 strains there is a wzz gene which controls the chain length of K(LPS) but has no effect on the O8 or O9 antigen.
Regulation of a heat shock sigma32 homolog in Caulobacter crescentus
Authors: Reisenauer A , Mohr C D , Shapiro L
Published: November 2016

High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase sigma32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli sigma32 and cross-reacts with a monoclonal antibody to E. coli sigma32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in sigma32 levels after a temperature shift from 30 to 42 degrees C similar to that described for E. coli. In addition, increasing the cellular content of sigma32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30 degrees C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and sigma32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to sigma32 expression in this organism. Both the rpoH gene and sigma32 protein were expressed constitutively throughout the cell cycle at 30 degrees C. The isolation of rpoH provides an important tool for future studies of the role of sigma32 in the normal physiology of C. crescentus.
LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family
Authors: Schnetz K , Stülke J , Gertz S , Krüger S , Krieg M , Hecker M , Rak B
Published: November 2016

Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.
Amino acids induce expression of BAP2, a branched-chain amino acid permease gene in Saccharomyces cerevisiae
Authors: Didion T , Grauslund M , Kielland-Brandt M C , Andersen H A
Published: November 2016

Branched-chain amino acid uptake in Saccharomyces cerevisiae is mediated by at least three transport systems: the general amino acid permease Gap1p, the branched-chain amino acid permease Bap2p, and one or more so far unknown permeases. Regulation of the transcription of BAP2 is mainly subject to the presence of certain amino acids in the medium. The level of transcription is low during growth on a minimal medium with proline as the sole nitrogen source. As assayed with a lacZ fusion, the level of transcription is slightly higher (3-fold) on a minimal medium with ammonium ions as a nitrogen source, and transcription is induced about 20-fold by addition of leucine (0.2 mM). As little as 10 microM leucine causes a fivefold induction. Addition of (L)-leucine to minimal proline medium, on the other hand, has no effect on BAP2 transcription. The two known permeases for transport of branched-chain amino acids, Gap1p and Bap2p, are thus not active at the same time. The BAP2 promoter contains one or two putative Gcn4p binding sites and one putative Leu3p binding site. None of the three is needed for induction by leucine. Induction of BAP2 transcription by leucine is accompanied by an increase in branched-chain amino acid uptake. This elevation is interpreted to be partly the result of an increased level of the Bap2p permease in the plasma membrane, because deletion of BAP2 slightly decreases the induction of uptake. There is still a leucine-inducible increase in branched-chain amino acid uptake in a delta gap1 delta bap2 strain, indicating that BAP2 shares leucine induction with at least one remaining branched-chain amino acid-transporting permease.
Phenotypes of Bacillus subtilis mutants lacking multiple class A high-molecular-weight penicillin-binding proteins
Authors: Popham D L , Setlow P
Published: November 2016

Examination of Bacillus subtilis strains containing multiple mutations affecting the class A high-molecular-weight penicillin-binding proteins (PBPs) 1, 2c, and 4 revealed a significant degree of redundancy in the functions of these three proteins. In rich media, loss of PBPs 2c and 4 resulted in no obvious phenotype. The slight growth and cell morphology defects associated with loss of PBP 1 were exacerbated by the additional loss of PBP 4 but not PBP 2c. Loss of all three of these PBPs slowed growth even further. In minimal medium, loss of PBPs 2c and 4 resulted in a slight growth defect. The decrease in growth rate caused by loss of PBP 1 was accentuated slightly by loss of PBP 2c and greatly by loss of PBP 4. Again, a lack of all three of these PBPs resulted in the slowest growth. Loss of PBP 1 resulted in a 22% reduction in the cell radius. Cultures of a strain lacking PBP 1 also contained some cells that were significantly longer than those produced by the wild type, and some of the rod-shaped cells appeared slightly bent. The additional loss of PBP 4 increased the number of longer cells in the culture. Slow growth caused by a mutation in prfA, a gene found in an operon with the gene encoding PBP 1, was unaffected by the additional loss of PBPs 2c and 4, whereas loss of both prfA and PBP 1 resulted in extremely slow growth and the production of highly bent cells.
Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus
Authors: Sau S , Lee C Y
Published: November 2016

Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.
The htpG gene of Bacillus subtilis belongs to class III heat shock genes and is under negative control
Authors: Schulz A , Schwab S , Homuth G , Versteeg S , Schumann W
Published: November 2016

We show that the htpG gene of Bacillus subtilis is induced by heat, as has been reported for the Escherichia coli homolog. Analysis of different mutants revealed that the htpG gene belongs to class III heat shock genes in B. subtilis. An about 10-fold induction after thermal upshock was found at the levels of both transcription and translation, and this induction resulted from enhanced synthesis of mRNA. By primer extension, we identified one potential transcription start site immediately downstream of a putative sigmaA-dependent promoter which became activated after thermal upshift. Northern blot analysis revealed that htpG is part of a monocistronic transcriptional unit. An operon fusion where the complete region between htpG and its upstream gene was fused to the bgaB reporter gene accurately reflected htpG expression. Analysis of this fusion revealed that, in contrast to other class III heat shock genes, htpG was not induced by osmotic upshock, by ethanol, or by oxygen limitation, suggesting that it belongs to a subgroup within class III. Deletion of the region upstream of the putative promoter resulted in an enhanced basal level of htpG expression, but the 10-fold induction was retained, suggesting that the upstream sequences are involved in the regulation of expression in the absence of heat shock.
Paraquat regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12 is SoxRS independent but modulated by sigma S
Authors: Membrillo-Hernández J , Kim S O , Cook G M , Poole R K
Published: November 2016

We report the first example of a gene, hmp, encoding a soluble flavohemoglobin in Escherichia coli K-12, which is up-regulated by paraquat in a SoxRS-independent manner. Unlike what is found for other paraquat-inducible genes, high concentrations of paraquat (200 microM) were required to increase the level of hmp expression, and maximal induction was observed only after 20 min of exposure to paraquat. Neither a mutation in soxS nor one in soxR prevented the paraquat-dependent increase in phi(hmp-lacZ) expression, but either mutant allele delayed full expression of phi(hmp-lacZ) activity after paraquat addition. Induction of hmp by paraquat was demonstrated in aerobically grown cultures during exponential growth and the stationary phase, thus revealing two Sox-independent regulatory mechanisms. Induction of hmp by paraquat in the stationary phase was dependent on the global regulator of stationary-phase gene expression, RpoS (sigma S). However, a mutation in rpoS did not prevent an increase in hmp expression by paraquat in exponentially growing cells. Induction of sigma S in the exponential phase by heat shock also induced phi(hmp-lacZ) expression in the presence of paraquat, supporting the role of sigma S in one of the regulatory mechanisms. Mutations in oxyR or rob, known regulators of several stress promoters in E. coli, had no effect on the induction of hmp by paraquat. Other known superoxide-generating agents (plumbagin, menadione, and phenazine methosulfate) were not effective in inducing hmp expression.
A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis
Authors: Raux E , Thermes C , Heathcote P , Rambach A , Warren M J
Published: November 2016

The role of cbiK, a gene found encoded within the Salmonella typhimurium cob operon, has been investigated by studying its in vivo function in Escherichia coli. First, it was found that cbiK is not required for cobalamin biosynthesis in the presence of a genomic cysG gene (encoding siroheme synthase) background. Second, in the absence of a genomic cysG gene, cobalamin biosynthesis in E. coli was found to be dependent upon the presence of cobA(P. denitrificans) (encoding the uroporphyrinogen III methyltransferase from Pseudomonas denitrificans) and cbiK. Third, complementation of the cysteine auxotrophy of the E. coli cysG deletion strain 302delta a could be attained by the combined presence of cobA(P. denitrificans) and the S. typhimurium cbiK gene. Collectively these results suggest that CbiK can function in fashion analogous to that of the N-terminal domain of CysG (CysG(B)), which catalyzes the final two steps in siroheme synthesis, i.e., NAD-dependent dehydrogenation of precorrin-2 to sirohydrochlorin and ferrochelation. Thus, phenotypically CysG(B) and CbiK have very similar properties in vivo, although the two proteins do not have any sequence similarity. In comparison to CysG, CbiK appears to have a greater affinity for Co2+ than for Fe2+, and it is likely that cbiK encodes an enzyme whose primary role is that of a cobalt chelatase in corrin biosynthesis.
Genome structure and phylogeny in the genus Brucella
Authors: Michaux-Charachon S , Bourg G , Jumas-Bilak E , Guigue-Talet P , Allardet-Servent A , O'Callaghan D , Ramuz M
Published: November 2016

PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb Spel fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.
Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11
Authors: Turner M S , Timms P , Hafner L M , Giffard P M
Published: November 2016

Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.
A novel resistance mechanism against beta-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyltransferase
Authors: Grebe T , Paik J , Hakenbeck R
Published: November 2016

Piperacillin resistance in Streptococcus pneumoniae was mediated by mutations in a novel gene, cpoA, that also confer transformation deficiency and a decrease in penicillin-binding protein la. cpoA is part of an operon located downstream of the primary sigma factor of S. pneumoniae. The deduced protein, CpoA, and the peptide encoded by the adjacent 3' open reading frame contained domains homologous to glycosyltransferases of procaryotes and eucaryotes that act on membrane-associated substrates, such as enzymes functioning in lipopolysaccharide core biosynthesis of gram-negative bacteria, RodD of Bacillus subtilis, which is involved in teichoic acid biosynthesis, and the human PIG-A protein, which is required for early steps of glycosylphosphatidylinositol anchor biosynthesis. This suggests that the cpo operon has a similar function related to cell surface components.
Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli
Authors: Sanders O I , Rensing C , Kuroda M , Mitra B , Rosen B P
Published: November 2016

In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated. Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF. The mutant was shown to be defective in polyol transport by GlpF. These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.
A virulent isolate of Salmonella enteritidis produces a Salmonella typhi-like lipopolysaccharide
Authors: Rahman M M , Guard-Petter J , Carlson R W
Published: November 2016

The lipopolysaccharide (LPS) of Salmonella enteritidis has been implicated as a virulence factor of this organism. Therefore, the LPS from a stable virulent isolate, SE6-E21, was compared with that from an avirulent isolate, SE6-E5. The LPSs were extracted, and the high-molecular-weight (HMW) LPS was separated from the low-molecular-weight (LMW) LPS for both isolates. Both the HMW and LMW LPSs were characterized by glycosyl composition and linkage analyses. Immunochemical characterization was performed by Western blotting using factor 9 antiserum and using S. typhimurium antiserum which contains factors 1, 4, 5, and 12(2). In addition, the polysaccharides released by mild acid hydrolysis were isolated and subjected to hydrolysis by bacteriophage P22, which contains endorhamnosidase activity. The resulting oligosaccharides were purified by using Bio-Gel P4 gel permeation chromatography and characterized by nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry (FAB-MS), tandem MS-MS, and matrix-assisted laser desorption time of flight MS. The results show that the HMW LPS O-antigen polysaccharides from both isolates are comprised of two different repeating units, -[-->2)-[alpha-Tyvp-(1-->3)]beta-D-Manp-(1-->4)-alpha-L-R hap-(1-->3)-alpha-D-Galp-(1-->]- (structure I) and [-->2)-[alpha-Tyvp-(1-->3)]beta-D-Manp-(1-->4)-alpha--L-R hap-(1-->3)-[alpha-D-Glcp-(1-->4)]alpha-D-Galp-(1-->]- (structure II). The LMW LPSs from both isolates contains truncated O-antigen polysaccharide which is comprised of only structure I. In the virulent SE6-E21 isolate, the HMW LPS has a structure I/II ratio of 1:1, while in the avirulent SE6-E5 isolate, this ratio is 7:1. While the 7:1 ratio represents the published level of glucosylation for S. enteritidis LPS as well as for S. enteritidis LPS purchased from Sigma Chemical Co., the 1:1 ratio found for the virulent SE6-E21 is identical to the high level of glucosylation reported for S. typhi LPS. Thus, the LPS from the virulent SE6-E21 isolate produces an S. typhi-like LPS. Furthermore, the amount of O-antigen polysaccharide in SE6-E21 was twice that in SE6-E5.
Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation
Authors: Atlung T , Knudsen K , Heerfordt L , Brøndsted L
Published: November 2016

The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons.
Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis
Authors: Chopade B A , Shankar S , Sundin G W , Mukhopadhyay S , Chakrabarty A M
Published: November 2016

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.