bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of FlgM in sigma D-dependent gene expression in Bacillus subtilis
Authors: Caramori T , Barilla D , Nessi C , Sacchi L , Galizzi A
Published: November 2016

The alternative sigma factor sigma D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis (sigmaD regulon). The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex. We have characterized the effects of an in-frame deletion mutation of flgM. By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different sigmaD-dependent promoters, i.e., Phag, PmotAB, and PfliDST. The number of flagellar filaments was only slightly increased by the flgM mutation. Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter. On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.
Effect of CO2 on the fermentation capacities of the acetogen Peptostreptococcus productus U-1
Authors: Misoph M , Drake H L
Published: November 2016

The fermentative capacities of the acetogenic bacterium Peptostreptococcus productus U-1 (ATCC 35244) were examined. Although acetate was formed from all the substrates tested, additional products were produced in response to CO2 limitation. Under CO2-limited conditions, fructose-dependent growth yielded high levels of lactate as a reduced end product; lactate was also produced under CO2-enriched conditions when fructose concentrations were elevated. In the absence of supplemental CO2, xylose-dependent growth yielded lactate and succinate as major reduced end products. Although supplemental CO2 and acetogenesis stimulated cell yields on fructose, xylose-dependent cell yields were decreased in response to CO2 and acetogenesis. In contrast, glycerol-dependent growth yielded high levels of ethanol in the absence of supplemental CO2, and pyruvate was subject to only acetogenic utilization independent of CO2. CO2 pulsing during the growth of CO2-limited fructose cultures stopped lactate synthesis immediately, indicating that CO2-limited cells were nonetheless metabolically poised to respond quickly to exogenous CO2. Resting cells that were cultivated at the expense of fructose without supplemental CO2 readily consumed fructose in the absence of exogenous CO2 and formed only lactate. Although the specific activity of lactate dehydrogenase was not appreciably influenced by supplemental C02 during cultivation, cells cultivated on fructose under CO2-enriched conditions displayed minimal capacities to consume fructose in the absence of exogenous CO2. These results demonstrate that the utilization of alternative fermentations for the conservation of energy and growth of P. productus U-1 is augmented by the relative availability of CO2 and growth substrate.
Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum
Authors: You Y , Elmore S , Colton L L , Mackenzie C , Stoops J K , Weinstock G M , Norris S J
Published: November 2016

Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.
Thermoregulation of kpsF, the first region 1 gene in the kps locus for polysialic acid biosynthesis in Escherichia coli K1
Authors: Cieslewicz M , Vimr E
Published: November 2016

The kps locus for biosynthesis of the capsular polysialic acid virulence factor in Escherichia coli K1 contains at least two convergently transcribed operons, designated region 1 and regions 2 plus 3. On the basis of DNA sequence analysis, kpsF appeared to be a good candidate for the first gene of region 1 (M. J. Cieslewicz, S. M. Steenbergen, and E. R. Vimr, J. Bacteriol. 175:8018-8023, 1993). A preliminary indication that kpsF is required for capsule production is the capsule-negative phenotype of an aph T insertion in the chromosomal copy of kpsF. The present communication describes the isolation and phenotypic characterization of this mutant. Although transcription through kpsF was required for capsule production, complementation analysis failed to indicate a clear requirement for the KpsF polypeptide. However, since E. coli contains at least two other open reading frames that could code for homologs of KpsF, the apparent dispensability of KpsF remains provisional. DNA sequence analysis of 1,100 bp upstream from the kpsF translational start site did not reveal any open reading frames longer than 174 nucleotides, consistent with kpsF being the first gene of region 1. Since kpsF appeared to be the first gene of a region whose gene products are required for polysialic acid transport and because capsule production is known to be thermoregulated, primer extension analyses were carried out with total RNA isolated from cells grown at permissive (37 degrees C) and nonpermissive (20 degrees C) temperatures. The results revealed a potentially complex kpsF promoter-like region that was transcriptionally silent at the nonpermissive temperature, suggesting that thermoregulation of region 1 may be exerted through variations in kpsF expression. Additional evidence supporting this conclusion was obtained by demonstrating the effects of temperature on expression of the gene kpsE, immediately downstream of kpsF. Chloramphenicol acetyltransferase assays were carried out with constructs containing the kpsF 5' untranslated region fused to a promoterless cat cassette, providing further evidence that kpsF is thermoregulated. Although the function of KpsF is unclear, primary structure analysis indicated two motifs commonly observed in regulatory proteins and homology with glucosamine synthase from Rhizobium meliloti.
Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system
Authors: Brian P , Riggle P J , Santos R A , Champness W C
Published: November 2016

Streptomycete antibiotic synthesis is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates. Streptomyces coelicolor produces several structurally and genetically distinct antibiotics. The S. coelicolor absA locus was defined by four UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation. We show that the absA locus encodes a putative eubacterial two-component sensor kinase-response regulator system. All four mutations lie within a single open reading frame, designated absA1, which is predicted to encode a sensor histidine kinase. A second gene downstream of absA1, absA2, is predicted to encode the cognate response regulator. In marked contrast to the antibiotic-deficient phenotype of the previously described absA mutants, the phenotype caused by disruption mutations in the absA locus is precocious hyperproduction of the antibiotics actinorhodin and undecylprodigiosin. Precocious hyperproduction of these antibiotics is correlated with premature expression of XylE activity in a transcriptional fusion to an actinorhodin biosynthetic gene. We propose that the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S. coelicolor.
Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase
Authors: Le Saux O , Gonzales T , Robert-Baudouy J
Published: November 2016

On the basis of chemical inhibition studies and a multiple alignment of four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seven conserved residues of the Pseudomonas fluorescens Pcp, which might be important for enzyme activity, have been modified by site-directed mutagenesis experiments. Wild-type and mutant Pcps were expressed in Escherichia coli, purified, and characterized by the ability to cleave the synthetic chromogenic substrate pyroglutamyl-beta-naphthylamide and the dipeptide pyroglutamyl-alanine. Substitution of Glu-10 and Glu-22 by Gln led to enzymes which displayed catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to those of the wild-type Pcp. These residues are not essential for the catalytic activity. Replacement of Asp-89 by Asn and Ala resulted in enzymes which retained nearly 25% of activity and which had no activity, respectively. Substitution of the Cys-144 and His-166 residues by Ala and Ser, respectively, resulted in inactive enzymes. Proteins with changes of Glu-81 to Gln and Asp-94 to Asn were not detectable in crude extract and were probably unstable in bacteria. Our results are consistent with the proposal that Cys-144 and His-166 constitute the nucleophilic and imidazole residues of the Pcp active site, while residue Glu-81, Asp-89, or Asp-94 might constitute the third part of the active site. These results lead us to propose Pcps as a new class of thiol aminopeptidases.
Molecular cloning and site-specific mutagenesis of a gene involved in arylsulfatase production in Campylobacter jejuni
Authors: Yao R , Guerry P
Published: November 2016

The arylsulfatase gene from Campylobacter jejuni 81-176 encodes a predicted protein of 69,293 Da which shows no sequence similarity with other known arylsulfatases. The gene hybridizes to other Ast+ strains of C. jejuni and Campylobacter sputorum subsp. bubulus, as well as to many Ast- strains of C. jejuni.
Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis
Authors: Brooke J S , Valvano M A
Published: November 2016

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.
Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 degrees C
Authors: Makin S A , Beveridge T J
Published: November 2016

Most Pseudomonas aeruginosa strains are able to produce two distinct lipopolysaccharide (LPS) O-polysaccharide types, A-band (common-antigen) and B-band (serotype-specific) LPSs. The relative expression levels of these two LPS types in P. aeruginosa PAO1 (O5 serotype) at various growth temperatures were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining or Western blotting (immunoblotting) with monoclonal antibodies specific for each O polysaccharide. A-band and B-band LPSs were expressed concurrently when the cells grew at 15, 25, and 35 degrees C; however, growth at 45 degrees C resulted in a surface deficiency in B-band LPS as determined by immunoblotting and agglutination with B-band-specific monoclonal antibody. Transfer of these cells (expressing A-band LPS but deficient in B-band LPS) [A+B-]) to a lower temperature (at which the division time was comparable) resulted in a rapid resumption of normal A-band and B-band expression. B-band LPS was detectable by immunoblotting before measurable growth of the culture had occurred.
Sequence of the bchG gene from Chloroflexus aurantiacus: relationship between chlorophyll synthase and other polyprenyltransferases
Authors: Lopez J C , Ryan S , Blankenship R E
Published: November 2016

The sequence of the Chloroflexus aurantiacus open reading frame thought to be the C. aurantiacus homolog of the Rhodobacter capsulatus bchG gene is reported. The BchG gene product catalyzes esterification of bacteriochlorophyllide a by geranylgeraniol-PPi during bacteriochlorophyll a biosynthesis. Homologs from Arabidopsis thaliana, Synechocystis sp. strain PCC6803, and C. aurantiacus were identified in database searches. Profile analysis identified three related polyprenyltransferase enzymes which attach an aliphatic alcohol PPi to an aromatic substrate. This suggests a broader relationship between chlorophyll synthases and other polyprenyltransferases.
Isolation and characterization of a gene from Streptomyces sp. strain C5 that confers the ability to convert daunomycin to doxorubicin on Streptomyces lividans TK24
Authors: Dickens M L , Strohl W R
Published: November 2016

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located between the daunomycin polyketide biosynthesis gene cluster and a dnrI (transcriptional activator) homolog, revealed the presence of a gene encoding a P-450-like enzyme with a deduced Mr of 46,096. Expression of this gene, named herein doxA, in Streptomyces lividans TY24 resulted in in vivo bioconversion of daunomycin to doxorubicin. DoxA showed specificity for only daunomycin and 13-dihydrodaunomycin, both of which were converted to doxorubicin. Daunomycinone (daunomycin aglycone), carminomycin, 13-dihydrocarminomycin, idarubicin, and aklavin were not apparent substrates for DoxA. In vector controls or in vectors in which doxA was poorly expressed, S. lividans catalyzed the reduction of daunomycin and other 13-oxo-anthracyclines and -anthracyclinones to their 13-dihydro homologs.
Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea
Authors: Ruggero D , Londei P
Published: November 2016

Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.
Phenotypic differentiation of "smart" versus "naive" bacteriophages of Bacillus subtilis
Authors: McVeigh R R , Yasbin R E
Published: November 2016

The temperate bacteriophages of Bacillus subtilis differ dramatically in their response to the induction of the SOS system during the development of competence and following DNA damage. While all temperate bacteriophages are induced following DNA damage, the "naive" bacteriophages (i.e., phi105 and SPO2) are also induced during the development of competence. On the other hand, "smart" bacteriophages (i.e., phi3T and SPbeta) are not induced during the development of competence, and furthermore, once competence has developed, these prophages can no longer be induced by DNA damage.
Fancy meeting you here! A fresh look at "prokaryotic" protein phosphorylation
Authors: Kennelly P J , Potts M
Published: November 2016

Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig. 1). As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific. Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes. Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought. Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc. The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.
Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes
Authors: Stuible H P , Wagner C , Andreou I , Huter G , Haselmann J , Schweizer E
Published: November 2016

The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid.
Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes
Authors: Morbidoni H R , de Mendoza D , Cronan J E
Published: November 2016

A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome.
Isolation and nucleotide sequence of the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase gene from Acetobacter xylinum
Authors: Petroni E A , Ielpi L
Published: November 2016

A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to be indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2.3-kb fragment of A. xylinum chromosomal DNA. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase enzyme, which is responsible for the transfer of an alpha-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial alpha-mannosyltransferases have a short COOH-terminal amino acid sequence in common.
The role of the 5'-end untranslated region of the mRNA for CspA, the major cold-shock protein of Escherichia coli, in cold-shock adaptation
Authors: Jiang W , Fang L , Inouye M
Published: November 2016

During cellular adaptation to low temperature, Escherichia coli transiently synthesizes the major cold-shock protein CspA. It was found that adaptation to cold shock is blocked when the 143-base sequence of the 5' untranslated region (5' UTR) of the cspA mRNA is overproduced. The overproduction of this UTR at 15 degrees C caused the synthesis of not only CspA but also other cold-shock proteins such as CspB and CsdA to be no longer transient but rather prolonged. In addition, inhibition of both the synthesis of cellular proteins other than cold-shock proteins and cell growth was observed. Interestingly, when CspA was also overproduced together with the 5' UTR, normal cold-shock adaptive response was resumed without a prolonged lag period of cell growth. This indicates that the 5' UTR of the cspA mRNA and its gene product CspA play a critical role in the regulation of the expression of cold-shock genes and cold-shock adaptation. An 11-base common sequence (cold box) was found in the 5' UTRs of cspA, cspB, and csdA mRNAs. Indeed, the 25-base sequence within the 5' UTR of the cspA mRNA containing the cold-box sequence was able to prolong CspA production at 15 degrees C. We propose that a putative repressor binds to the cold-box sequence of the cold-shock mRNAs during the adaptive process and this binding in turn blocks the transcription of the cold-shock genes or destabilizes their mRNAs. CspA appears to promote either directly or indirectly the repressor function.
Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1
Authors: Climo M W , Sharma V K , Archer G L
Published: November 2016

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.
SpoIIE mutants of Bacillus subtilis comprise two distinct phenotypic classes consistent with a dual functional role for the SpoIIE protein
Authors: Barák I , Youngman P
Published: November 2016

Mutations in the spoIIE locus of Bacillus subtilis block sporulation at the stage of asymmetric septation and prevent compartment-specific activation of the transcription factor delta F. Recent ultrastructural studies of spoIIE mutants led to the conclusion that cells blocked at the stage of asymmetric septation form primarily thick septal structures similar to those formed at the mid-cell site during vegetative growth, although in an earlier study Piggot (J. Bacteriol. 114:1241-1253, 1973) clearly detected a more complex range of phenotypes. We have examined the phenotypes of six spoIIE mutants, including one example of the previously studied null type, spoIIE21. We confirmed that the spoIIE21 mutant and two other null mutants exhibit the classic thick-septum phenotype. However, two of the missense mutants, the spoIIE64 and spoIIE71 mutants, were found to display a strikingly different phenotype characterized by the presence of only thin asymmetric septa, frequently at both polar positions, as noted by Piggot. This phenotype is essentially identical to those of spoIIA (delta F) and spoIIG (delta E) null mutants, which also form sporulation septa that appear structurally normal at the level of electron microscopy. Despite the formation of apparently normal asymmetric septa, spoIIE64 and spoIIE71 mutants are fully defective in activation of delta F-dependent gene expression. These results indicate that the functional roles performed by SpoIIE in septum assembly and sigma factor regulation are distinct and separable.
Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis
Authors: Schurr M J , Yu H , Martinez-Salazar J M , Boucher J C , Deretic V
Published: November 2016

The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.
Escherichia coli DNA repair genes radA and sms are the same gene
Authors: Song Y , Sargentini N J
Published: November 2016

Escherichia coli strains carrying radA100 or sms mutations were identical in their sensitivities to either methyl methanesulfonate or UV radiation treatment and in their plasmid complementation patterns for UV radiation survival. DNA sequencing analysis of the radA mutant and radA+ strains and comparison of their sequences with the published sms gene sequence showed the radA mutant to differ only by a G-to-A transition mutation, which is predicted to change a cysteine in a zinc-finger motif to tyrosine. The sms gene is concluded to be identical to the previously described radA gene.
A developmental stage-specific histone H1 homolog of Coxiella burnetii
Authors: Heinzen R A , Hackstadt T
Published: November 2016

Two DNA-binding proteins have been detected in Coxiella burnetii by southwestern (DNA-protein) blotting. One of these, termed Hq1, is enriched in the small cell variant stage of the developmental cycle and displays compositional and primary amino acid sequence similarities to eukaryotic histone H1. C. burnetii appears to be another example of an intracellular parasite with morphologically distinct developmental forms whose nucleoid structure may be controlled by histone H1 homologs.
Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD
Authors: Kappes R M , Kempf B , Bremer E
Published: November 2016

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.
Interaction between FtsZ and inhibitors of cell division
Authors: Huang J , Cao C , Lutkenhaus J
Published: November 2016

The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system. An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains. This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA. No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system. However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD. The implications of these findings for cell division are discussed.
Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes
Authors: Soncini F C , García Véscovi E , Solomon F , Groisman E A
Published: November 2016

The PhoP-PhoQ two-component system is essential for virulence in Salmonella typhimurium. This system controls expression of some 40 different proteins, yet most PhoP-regulated genes remain unknown. To identify PhoP-regulated genes, we isolated a library of 50,000 independent lac gene transcriptional fusion strains and investigated whether production of beta-galactosidase was regulated by PhoP. We recovered 47 lac gene fusions that were activated and 7 that were repressed when PhoP was expressed. Analysis of 40 such fusions defined some 30 loci, including mgtA and mgtCB, which encode two of the three Mg2+ uptake systems of S. typhimurium; ugd, encoding UDP-glucose dehydrogenase; phoP, indicative that the phoPQ operon is autoregulated; and an open reading frame encoding a protein with sequence similarity to VanX, a dipeptidase required for resistance to vancomycin. Transcription of PhoP-activated genes was regulated by the levels of Mg2+ in a PhoP-dependent manner. Strains with mutations in phoP or phoQ were defective for growth in low-Mg2+ media. The mgtA and mgtCB mutants reached lower optical densities than the wild-type strain in low-Mg2+ liquid media but displayed normal growth on low-Mg2+ solid media. Six PhoP-activated genes were identified as essential to form colonies on low-Mg'+ solid media. Cumulatively, our experiments establish that the PhoP-PhoQ system governs the adaptation to magnesium-limiting environments.
Identification of sigma S-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma S and cyclic AMP receptor protein
Authors: Fang F C , Chen C Y , Guiney D G , Xu Y
Published: November 2016

sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation. Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth. sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds. Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci. Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase. For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth. The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors.
The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus
Authors: Elsen S , Colbeau A , Chabert J , Vignais P M
Published: November 2016

The hupT, hupU, and hupV genes, which are located upstream from the hupSLC and hypF genes in the chromosome of Rhodobacter capsulatus, form the hupTUV operon expressed from the hupT promoter. The hupU and hupV genes, previously thought to belong to a single open reading frame, encode HupU, of 34.5 kDa (332 amino acids), and HupV, of 50.4 kDa (476 amino acids), which are >/= 50% identical to the homologous Bradyrhizobium japonicum HupU and HupV proteins and Rhodobacter sphaeroides HupU1 and HupU2 proteins, respectively; they also have 20 and 29% similarity with the small subunit (HupS) and the large subunit (HupL), respectively, of R. capsulatus [NiFe]hydrogenase. HupU lacks the signal peptide of HupS and HupV lacks the C-terminal sequence of HupL, which are cleaved during hydrogenase processing. Inactivation of hupV by insertional mutagenesis or of hupUV by in-frame deletion led to HupV- and Hup(UV)- mutants derepressed for hydrogenase synthesis, particularly in the presence of oxygen. These mutants were complemented in trans by plasmid-borne hupTUV but not by hupT or by hupUV, except when expressed from the inducible fru promoter. Complementation of the HupV- and Hup(UV)- mutants brought about a decrease in hydrogenase activity up to 10-fold, to the level of the wild-type strain B10, indicating that HupU and HupV participate in negative regulation of hydrogenase expression in concert with HupT, a sensor histidine kinase involved in the repression process. Plasmid-borne gene fusions used to monitor hupTUV expression indicated that the operon is expressed at a low level (50- to 100-fold lower than hupS).
A positive control mutant of the transcription activator protein FIS
Authors: Gosink K K , Gaal T , Bokal A J , Gourse R L
Published: November 2016

The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.
Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species
Authors: Kelly R F , Whitfield C
Published: November 2016

Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7%. The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events.
Quorum sensing in Vibrio fischeri: evidence that S-adenosylmethionine is the amino acid substrate for autoinducer synthesis
Authors: Hanzelka B L , Greenberg E P
Published: November 2016

Synthesis of the autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI. The autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis. We have measured autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid. Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for autoinducer synthesis. The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent autoinducer synthesis. Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for autoinducer synthesis. The amount of 15N-labeled methionine incorporated into the autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry. The labeling studies show that even in the presence of homoserine, almost all of the autoinducer produced contains the 15N label from methionine. Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V. fischeri autoinducer.
Dynamic aspects of colicin N translocation through the Escherichia coli outer membrane
Authors: El Kouhen R , Pagès J M
Published: November 2016

Colicin N is a bacteriocin that kills sensitive Escherichia coli cells. After binding to the cell surface-exposed receptor, a short period exists when a significant number of the cell-associated colicin N molecules are sensitive to external enzymes. Two colicin N populations are discriminated by proteases: the susceptible pool bound to OmpF porin on the cell surface and another population corresponding to protease-inaccessible colicin N. During translocation, colicin N reaches the periplasmic space and proteolytic cleavage of the colicin occurs only when the outer membrane barrier is permeabilized.
Purification and activation properties of UreD-UreF-urease apoprotein complexes
Authors: Moncrief M B , Hausinger R P
Published: November 2016

In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S. Park, M.B. Carr, and R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S. Park and R.P. Hausinger, J. Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein. These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2. The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins. Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand.
Reactivation of the Bacillus subtilis anti-sigma B antagonist, RsbV, by stress- or starvation-induced phosphatase activities
Authors: Voelker U , Voelker A , Haldenwang W G
Published: November 2016

sigma B is a secondary sigma factor that controls the general stress regulon in Bacillus subtilis. The regulon is activated when sigma B is released from a complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate mechanisms cause sigma B release: an ATP-responsive mechanism that correlates with nutritional stress and an ATP-independent mechanism that responds to environmental insult (e.g., heat shock and ethanol treatment). ATP levels are thought to directly affect RsbW's binding preference. Low levels of ATP cause RsbW to release sigma B and bind to an alternative protein (RsbV), while high levels of ATP favor RsbW-sigma B complex formation and inactivation of RsbV by an RsbW-dependent phosphorylation. During growth, most of the RsbV is phosphorylated (RsbV-P) and inactive. Environmental stress induces the release of sigma B and the formation of the RsbW-RsbV complex, regardless of ATP levels. This pathway requires the products of additional genes encoded within the eight-gene operon (sigB) that includes the genes for sigma B, RsbW, and RsbV. By using isoelectric focusing techniques to distinguish RsbV from RsbV-P and chloramphenicol treatment or pulse-chase labeling to identify preexisting RsbV-P, we have now determined that stress induces the dephosphorylation of RsbV-P to reactivate RsbV. RsbV-P was also found to be dephosphorylated upon a drop in intracellular ATP levels. The stress-dependent and ATP-responsive dephosphorylations of RsbV-P differed in their requirements for the products of the first four genes (rsbR, -S, -T, and -U) of the sigB operon. Both dephosphorylation reactions required at least one of the genes included in a deletion that removed rsbR, -S, and -T; however, only an environmental insult required RsbU to reactivate RsbV.