bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli sigma 54 RNA polymerase recognizes Caulobacter crescentus flbG and flaN flagellar gene promoters in vitro
Authors: Ninfa A J , Mullin D A , Ramakrishnan G , Newton A
Published: November 2016

A set of the periodically regulated flagellar (fla) genes of Caulobacter crescentus contain conserved promoter sequence elements at -24 and -12 that are very similar to the sequence of the nitrogen assimilation (Ntr) and nitrogen fixation (Nif) promoters of enteric bacteria and Rhizobium spp. Transcription from Ntr and Nif promoters requires RNA polymerase containing sigma 54 instead of the usual sigma 70 and, in the case of the Ntr promoters, is activated by the transcription factors NRI and NRII. We have now demonstrated that the C. crescentus flbG and flaN promoters, which contain the Ntr/Nif type of consensus sequence, are utilized by purified Escherichia coli sigma 54 RNA polymerase (E sigma 54) in the presence of NRI and NRII but not by the purified sigma 70 RNA polymerase (E sigma 70) of E. coli. Oligonucleotide-generated flbG promoter deletions that removed the highly conserved GG dinucleotide at -24 or the GC dinucleotide at -12 or altered the spacing between the -24 and -12 sequence elements prevented utilization of the flbG promoter by the E. coli E sigma 54. Transversions of T to G at positions -26 and -15 also inactivated flbG promoter function in the E. coli cell-free transcription system, while a transition of G to A at position -16 in the nonconserved spacer region had no effect. The C. crescentus flaO and flbF promoters, which do not contain the Ntr/Nif-type promoter consensus sequence, were not utilized by either purified E sigma 54 or E sigma 70 from E. coli. Our results help to define the features of the Ntr/Nif-type consensus sequence required for promoter utilization by purified E. coli E sigma 54 and support the idea that C. crescentus may contain a specialized polymerase with similar promoter specificity required for expression of a set of fla genes.
Peptidoglycan-polysaccharide complex in the cell wall of the filamentous prochlorophyte Prochlorothrix hollandica
Authors: Jürgens U J , Burger-Wiersma T
Published: November 2016

A peptidoglycan-polysaccharide complex composed of N-acetylglucosamine, N-acetylmuramic acid, muramic acid 6-phosphate, L-alanine, D-alanine, D-glutamic acid, meso-diaminopimelic acid, N-acetylmannosamine, mannose, galactose, glucose, and phosphate was isolated from cell walls of the filamentous prochlorophyte Prochlorothrix hollandica; this complex was similar in chemical composition and structure to that found in cyanobacteria. Peptide patterns of partial acid hydrolysates of the isolated peptidoglycan revealed an A1 gamma structure with direct cross-linkage (m-diaminopimelic acid-D-alanine) of the peptide side chains. The degree of cross-linkage (63%) was found to be in the range of values obtained for gram-positive bacteria and cyanobacteria.
Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein
Authors: Dean D A , Fikes J D , Gehring K , Bassford P J , Nikaido H
Published: November 2016

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.
Gene replacement and retrieval with recombinant M13mp bacteriophages
Authors: Blum P , Holzschu D , Kwan H S , Riggs D , Artz S
Published: November 2016

We have developed an allele exchange system for shuttling sequences of DNA to and from their original chromosomal loci. Cloned segments of the histidine operon of Salmonella typhimurium and the lactose operon of Escherichia coli served as target sequences and were used to develop the system. Replacement and retrieval of target sequences used the phage M13mp vectors and proceeded through an M13 lysogen intermediate. The intermediates and products of allele exchange were characterized by genetic and hybridization analyses. Several unique properties of M13 lysogens were exploited to devise positive selections to detect integration and excision. These positive selections were used to manipulate phenotypically silent alleles.
Export-defective lamB protein is a target for translational control caused by ompC porin overexpression
Authors: Click E M , Schnaitman C A
Published: November 2016

Overexpression of OmpC protein from an inducible plasmid vector reduced the amount of the precursor form of LamB protein in LamB signal sequence mutants. The stability of the precursor form of LamB protein was not affected, indicating that the effect of OmpC overexpression was on the synthesis of the precursor rather than on degradation. These results indicate that a functional signal sequence is not required on an outer membrane protein for it to be a target for translational control.
Rhizobium leguminosarum CFN42 genetic regions encoding lipopolysaccharide structures essential for complete nodule development on bean plants
Authors: Cava J R , Elias P M , Turowski D A , Noel K D
Published: November 2016

Eight symbiotic mutants defective in lipopolysaccharide (LPS) synthesis were isolated from Rhizobium leguminosarum biovar phaseoli CFN42. These eight strains elicited small white nodules lacking infected cells when inoculated onto bean plants. The mutants had undetectable or greatly diminished amounts of the complete LPS (LPS I), whereas amounts of an LPS lacking the O antigen (LPS II) greatly increased. Apparent LPS bands that migrated between LPS I and LPS II on sodium dodecyl sulfate-polyacrylamide gels were detected in extracts of some of the mutants. The mutant strains were complemented to wild-type LPS I content and antigenicity by DNA from a cosmid library of the wild-type genome. Most of the mutations were clustered in two genetic regions; one mutation was located in a third region. Strains complemented by DNA from two of these regions produced healthy nitrogen-fixing nodules. Strains complemented to wild-type LPS content by the other genetic region induced nodules that exhibited little or no nitrogenase activity, although nodule development was obviously enhanced by the presence of this DNA. The results support the idea that complete LPS structures, in normal amounts, are necessary for infection thread development in bean plants.
Apparent minimal size required for cell division in Escherichia coli
Authors: Grossman N , Ron E Z
Published: November 2016

The experiments described in this report were designed to find out whether there is a minimal size threshold for cell division or for DNA replication in Escherichia coli. Cells with decreasing size (or mass) were obtained by successive amino acid starvations. Following two starvations, the cells were at least 30% smaller than unstarved newborn cells. The results suggest that this size is below a minimal size threshold for cell division but not for initiation of DNA replication.
Conservation between coding and regulatory elements of Rhizobium meliloti and Rhizobium leguminosarum dct genes
Authors: Jiang J , Gu B H , Albright L M , Nixon B T
Published: November 2016

Complementation of Rhizobium leguminosarum dct mutants with a cosmid bank yielded Rhizobium meliloti homologs of the dctA, dctB, and dctD genes. The genes dctB and dctD are thought to form a two-component system which responds to the presence of C4-dicarboxylates to regulate expression of a transport protein encoded by dctA. DNA sequence analysis showed that dct coding and intergenic regions, including putative binding sites for the dctD protein and sigma 54-RNA polymerase, were highly conserved between these two Rhizobium species. Mutation of R. meliloti dctD showed that it was not essential for symbiotic nitrogen fixation but was needed for growth on succinate and the expression of a dctA-lacZ fusion gene in free-living cells. Hybridization of R. meliloti genomic DNA with probes representing the central portion of dctD potentially identified more than 20 similar regulatory genes, all of which are likely to depend upon the alternative sigma factor encoded by rpoN and stimulate transcription in a manner very similar to ntrC activation of glnA in enteric bacteria.
Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair
Authors: Iwasaki H , Shiba T , Makino K , Nakata A , Shinagawa H
Published: November 2016

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.
Differential regulation by iron of regA and toxA transcript accumulation in Pseudomonas aeruginosa
Authors: Frank D W , Storey D G , Hindahl M S , Iglewski B H
Published: November 2016

Iron regulation of toxA and regA transcript accumulation was examined in Pseudomonas aeruginosa PA103 containing the regA gene on a multicopy plasmid. The patterns of transcript accumulation for toxA and regA were found to be positively correlated. Dot blot and Northern (RNA) blot analysis of total RNA isolated throughout the bacterial growth cycle indicated that multiple copies of the regA gene uncoupled iron repression of the first phase of transcript accumulation for both regA and toxA genes. However, regulation by iron of the second phase of transcript accumulation for each gene was unaffected by several regA gene copies. Total toxin production was increased in cells with multiple copies of regA grown in either low- or high-iron medium. Primer extension analysis of regA mRNA extracted from cells grown in high- and low-iron medium and examined at different points in the cell growth cycle supported the hypothesis that iron regulation of regA transcription occurs at the level of transcriptional initiation. Two start sites were shown for regA transcription at -164 and -75 base pairs from the ATG start codon. The differential regulation of regA transcript accumulation when regA is present in single or multiple copy and the mapping of independent start sites for regA mRNA support the evidence that regA transcription is directed by independently regulated promoter regions.
Construction of an Agrobacterium tumefaciens C58 recA mutant
Authors: Farrand S K , O'Morchoe S P , McCutchan J
Published: November 2016

Clones encoding the recA gene of Agrobacterium tumefaciens C58 were isolated from a cosmid bank by complementation of an Escherichia coli recA mutation. Subcloning and mutagenesis with the lacZ fusion transposon Tn3HoHo1 located the Agrobacterium recA gene to a 1.3-kilobase segment of DNA. beta-Galactosidase expression from the fusions established the direction in which the gene was transcribed. The gene restored homologous recombination as well as DNA repair functions in E. coli recA mutants. Similar complementation of DNA repair functions was observed in the UV-induced Rec- Agrobacterium mutant, LBA4301. The Agrobacterium recA gene was disrupted by insertion of a cassette encoding resistance to erythromycin, and the mutated gene was marker exchanged into the chromosome of strain NT-1. The resulting strain, called UIA143, was sensitive to UV irradiation and methanesulfonic acid methyl ester and unable to carry out homologous recombination functions. The mutation was stable and had no effect on other genetic properties of the Agrobacterium strain, including transformability and proficiency as a conjugal donor or recipient. Furthermore, strain UIA143 became tumorigenic upon introduction of a Ti plasmid, indicating that tumor induction is independent of recA functions. Sequence homology was detected between the recA genes of strain C58 and E. coli as well as with DNA isolated from agrobacteria representing the three major biochemically differentiated biovars of this genus. In some cases, biovar-specific restriction fragment length polymorphisms were apparent at the recA locus.
Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii
Authors: Jetten M S , Stams A J , Zehnder A J
Published: November 2016

In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.
Aromatic aminotransferase activity and indoleacetic acid production in Rhizobium meliloti
Authors: Kittell B L , Helinski D R , Ditta G S
Published: November 2016

Bacterial indoleacetic acid (IAA) production, which has been proposed to play a role in the Rhizobium-legume symbiosis, is a poorly understood process. Previous data have suggested that IAA biosynthesis in Rhizobium meliloti can occur through an indolepyruvate intermediate derived from tryptophan by an aminotransferase activity. To further examine this biosynthetic pathway, the aromatic aminotransferase (AAT) activity of Rhizobium meliloti 102F34 (F34) was characterized. At least four proteins were detected on nondenaturing gels of F34 protein extracts that exhibited AAT activity. All four of these AATs were constitutively produced and utilized the aromatic amino acids tryptophan, phenylalanine, and tyrosine as amino substrates. Two AATs were also capable of using aspartate. Plasmids from an F34 gene bank were identified that coded for the synthesis of at least three of these proteins, and the respective gene sequences were localized by transposon mutagenesis. Selected transposon insertions were recombined into the F34 genome to produce strains defective in two of these proteins (AAT1 and AAT2). Characterization of the mutants revealed that neither was essential for the biosynthesis of IAA in the absence of exogenous tryptophan, but that both contributed to IAA biosynthesis when high levels of exogenous tryptophan were present. AAT1 and AAT2 were also not required for the production of a minimal level of aromatic amino acids, but both were able to scavenge nitrogen from the aromatic amino acids during nitrogen deprivation. Neither AAT1 nor AAT2 was essential for symbiosis with alfalfa.
Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon
Authors: Furukawa K , Hayase N , Taira K , Tomizuka N
Published: November 2016

All the genes we examined that encoded biphenyl/polychlorinated biphenyl (PCB) degradation were chromosomal, unlike many other degradation-encoding genes, which are plasmid borne. The molecular relationship of genes coding for biphenyl/PCB catabolism in various biphenyl/PCB-degrading Pseudomonas, Achromobacter, Alcaligenes, Moraxella, and Arthrobacter strains was investigated. Among 15 strains tested, 5 Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on the XhoI 7.2-kilobase fragment corresponding to that of Pseudomonas pseudoalcaligenes KF707. More importantly, the restriction profiles of these XhoI 7.2-kilobase fragments containing bphABC genes were very similar, if not identical, despite the dissimilarity of the flanking chromosomal regions. Three other strains also possessed bphABC genes homologous with those of KF707, and five other strains showed weak or no significant genetic homology with bphABC of KF707. The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well to the DNA homology. On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1, lacked genetic as well as immunological homology with any of the other 15 biphenyl/PCB degraders tested. The existence of the nearly identical chromosomal genes among various strains may suggest that a segment containing the bphABC genes has a mechanism for transferring the gene from one strain to another.
DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters
Authors: Fisher R F , Long S R
Published: November 2016

The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family. They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer. NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes. We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent. NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters. The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1.
Restriction fragment length polymorphism analysis of Rhizobium galegae strains
Authors: Kaijalainen S , Lindström K
Published: November 2016

Total DNA of various Rhizobium galegae strains representing different geographical origins, and taxonomic divergence was digested with three restriction enzymes separately, Southern blotted, and hybridized with six heterologous probes. The sequence divergences for different pairwise comparisons were calculated from proportions of conserved hybridizing fragments. The unweighted pair group method was used to group the strains. The symbiotic common nod and nifHDK probes used were highly conserved and grouped the strains according to the host plant, Galega orientalis or G. officinalis. The grouping derived from combined data of the constitutive hemA, glnA, ntrC, and recA probes was similar to that obtained in total DNA-DNA hybridization experiments. The constitutive probes grouped the strains in a different order than did the symbiotic probes, a result that may reflect interstrain transfer of symbiotic sequences in the course of evolution.
Nucleotide sequences of dnaE, the gene for the polymerase subunit of DNA polymerase III in Salmonella typhimurium, and a variant that facilitates growth in the absence of another polymerase subunit
Authors: Lancy E D , Lifsics M R , Munson P , Maurer R
Published: November 2016

The dnaE gene of Salmonella typhimurium, like that of Escherichia coli, encodes the alpha subunit containing the polymerase activity of the principal replicative enzyme, DNA polymerase III. This gene, or one nearby, has been identified as the locus of suppressor mutations that promote growth by cells deleted for dnaQ, the gene for the editing subunit of this enzyme complex. Using a combination of nucleotide sequencing and marker rescue experiments, the alteration in one such suppressor was identified as a valine-to-glycine substitution at amino acid 832 of the 1,160-amino-acid alpha polypeptide. The alpha polypeptides of E. coli and S. typhimurium are identical in size and in 97% of their amino acid residues. Their identity includes the valine residue that was changed in the suppressor allele of S. typhimurium. We also localized a temperature-sensitive dnaE mutation to the 3' half of dnaE.
Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography
Authors: Leidenix M J , Jacoby G H , Henderson T A , Young K D
Published: November 2016

Membrane vesicles from the envelope of Escherichia coli were separated by electrophoresis through dilute agarose and by sizing chromatography through Sephacryl S-1000. These techniques revealed that proteins were associated with different subsets of vesicles. In particular, dilute agarose electrophoresis clearly separated the inner membrane penicillin-binding proteins (PBPs) into different vesicle groups. Vesicles containing PBPs 4, 6, 7, and 8 migrated rapidly through agarose; vesicles with PBPs 1a, 1b, 2, 3, and 5 eluted later. With the exception of PBP 4, which migrated with PBPs 1 through 5, chromatography through Sephacryl S-1000 was able to distinguish the same two vesicle sets, though the extent of separation was poorer than with agarose. The existence of these associations among vesicles and proteins suggests that there is an organization to the inner membrane of E. coli which is not observed when membrane vesicles are separated solely on the basis of density in sucrose gradients.
Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene
Authors: Owttrim G W , Coleman J R
Published: November 2016

The expression of the cyanobacterial recA gene, isolated from Anabaena variabilis, has been examined at the levels of transcript and protein abundance. Exposure of the cyanobacterium to a variety of DNA-damaging agents, including mitomycin C, methyl methanesulfonate, and UV irradiation, results in a rapid increase in the abundance of the recA transcript above basal levels as determined by Northern (RNA) blot analysis. A concomitant increase in the abundance of a 37- to 38-kilodalton polypeptide was also detected by Western (immuno-) blot analysis of soluble cyanobacterial polypeptides using polyclonal antiserum directed against the Escherichia coli recA protein. The cyanobacterial polypeptide is of the same molecular mass as that synthesized by an in vitro, DNA-directed procaryotic transcription-translation system primed with an A. variabilis genomic fragment containing the recA gene. Nucleotide sequence analysis of the cyanobacterial gene revealed a protein of 358 amino acids with a molecular weight of 38,403 daltons. The A. variabilis and E. coli recA genes share similarity at 58% of the amino acid residues; however, an E. coli-like lexA repressor-binding site is not present in the A. variabilis promoter region. The similarities of A. variabilis and E. coli recA expression and gene sequence are discussed.
Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154
Authors: Palissa H , von Döhren H , Kleinkauf H , Ting H H , Baldwin J E
Published: November 2016

The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level beta-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including alpha-aminoadipic acid and cysteine. The Km for alpha-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a beta-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of beta-lactams in bacteria.
Timing of spoII gene expression relative to septum formation during sporulation of Bacillus subtilis
Authors: Gholamhoseinian A , Piggot P J
Published: November 2016

spoII mutants formed heat-resistant spores when transformed with spo+ DNa near the start of sporulation. Many of the spores formed remained genetically spoII. It is deduced from this result and previous epistasis experiments that the spoII loci are transcribed before the spore septum is formed.
A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli
Authors: Spikes D , Setlow J K
Published: November 2016

The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation.
Crystalline cell surface layer of Mycobacterium bovis BCG
Authors: Lounatmaa K , Brander E
Published: November 2016

A paracrystalline surface layer (S layer) was found as the outermost layer of the cell wall of five Mycobacterium bovis BCG strains. An oblique arrangement of the subunits in the S layer was only clearly seen in thin-sectioned and shadowed preparations, and the unit constant was about 5.5 nm.
Escherichia coli K-12 cell-cell interactions seen by time-lapse video
Authors: Shapiro J A , Hsu C
Published: November 2016

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.
Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIP501
Authors: Krah E R , Macrina F L
Published: November 2016

pIP501 is a 30.2-kilobase (kb) broad-host-range conjugative streptococcal plasmid which encodes chloramphenicol and erythromycin resistance. A smaller conjugation-proficient derivative of pIP501 has been constructed and designated pVA1702. pVA1702 is 25.2 kb, encodes only kanamycin resistance, and confers conjugative ability in an Enterococcus faecalis mating system. The temperature-sensitive transposon delivery vectors pTV1ts and pTV32 carrying Tn917 and Tn917lac, respectively, were used to create insertion mutants of pVA1702 in E. faecalis. These insertions enabled us to localize the determinants conferring conjugation to two separate regions of pVA1702: the 7.5-kb region A and the 8.8-kb region B. A nested set of deletions of the 7.5-kb region was subcloned in Escherichia coli, and the expression of these deletions was examined in an E. coli minicell system. The genes for three conjugal proteins have been located and designated cnjA, cnjB, and cnjC (for conjugation). Two other peptides that are transcribed in a divergent direction from the cnj genes were encoded in the A region, but it is not known whether they are involved in conjugation. These studies indicate that the conjugation genes of pIP501 are arranged in multiple transcriptional units.
Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures
Authors: Bukau B , Walker G C
Published: November 2016

Major heat shock proteins, such as the Escherichia coli DnaK protein, not only are required for cell growth after heat shock but seem to possess important functions in cellular metabolism at normal growth temperatures as well. E. coli delta dnaK52 mutants have severe cellular defects at 30 degrees C, one of which is in cell division (B. Bukau and G. C. Walker, J. Bacteriol, 171:2337-2346, 1989). Here we show that at 30 degrees C, delta dnaK52 mutants have defects in chromosome segregation and in maintenance of low-copy-number plasmids. Fluorescence microscopic analysis revealed that chromosomes were frequently lacking at peripheries of cell filaments of delta dnaK52 mutants and clustered at other locations. In other parts of the cell filaments, chromosomes were apparently normally distributed and they were also present in most of the small cells found in populations of delta dnaK52 cells. These defects might be at the level of DNA replication, since delta dnaK52 mutants have a threshold lower rate of DNA synthesis than wild-type cells. Chromosome segregation defects of delta dnaK52 mutants were also observed in an rnh dnaA mutant background, in which initiation of DNA replication is DnaA-oriC independent. We also found that low-copy-number P1 miniplasmids could not be stably maintained in delta dnaK52 mutants at 30 degrees C. delta par P1 miniplasmids that carry the P1-encoded rep functions required for their replication but lack the P1-encoded par functions required for faithful partitioning of the plasmids during cell division were also unstable in delta dnaK52 mutants. Taken together, our results indicate important, although not absolutely essential, functions for DnaK at 30 degrees C in one or more processes necessary for correct replication and/or partitioning of chromosomes and P1 miniplasmids. Furthermore, we found that P1 miniplasmids were also highly unstable in dnaJ259 mutants, indicating a role for the DnaJ heat shock protein in maintenance of these plasmids.
Purification of a fourth glucosyltransferase from Streptococcus sobrinus
Authors: Yamashita Y , Hanada N , Takehara T
Published: November 2016

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.
Removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes, rebinding of CO dehydrogenase to depleted membranes, and restoration of respiratory activities
Authors: Jacobitz S , Meyer O
Published: November 2016

In Pseudomonas carboxydovorans, CO dehydrogenase and hydrogenase were found in association with the cytoplasmic membrane in a weakly bound and a tightly bound pool. The pools could be experimentally distinguished on the basis of resistance to removal by washes in low-ionic-strength buffer. The tightly bound pool of the enzymes could be differentially solubilized under conditions leaving the electron transport system intact and with the nondenaturing zwitterionic detergent 3-(3-cholamidopropyl) dimethylammonio 1-propane-sulfonic acid (CHAPS) and the nonionic detergent dodecyl beta-D-maltoside. In vitro reconstitution of depleted membranes with the corresponding supernatants containing CO dehydrogenase led to binding of the enzyme and to reactivation of respiratory activities with CO. The reconstitution reaction required cations with effectiveness which increased with increasing ionic charge: monovalent (Li+), divalent (Mg2+, Mn2+), or trivalent (Cr3+, La3+). Reconstitution of depleted membranes with CO dehydrogenase was specific for CO-grown bacteria. Cytoplasmic membranes from H2- or heterotrophically grown Pseudomonas carboxydovorans had no affinity for CO dehydrogenase at all, indicating the absence of the physiological electron acceptor of the enzyme, which presumably is cytochrome b561, or another membrane anchor.
Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775
Authors: Singer J T
Published: November 2016

The recA analog from Vibrio anguillarum 775 was isolated by complementation of recA mutations in Escherichia coli, and its protein product was identified. The recA analog promoted recombination between two partially deleted lactose operons, stimulated both spontaneous and mitomycin C-induced phage production in RecA- lambda lysogens, and restored near wild-type levels of resistance to UV radiation and methyl methanesulfonate.
Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies
Authors: Sussman R , Alexander H B
Published: November 2016

To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.
Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B
Authors: Deguchi Y , Yamato I , Anraku Y
Published: November 2016

Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.
Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus
Authors: Eberz G , Eitinger T , Friedrich B
Published: November 2016

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.
Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical
Authors: Walkup L K , Kogoma T
Published: November 2016

Two-dimensional gel analyses were made of proteins synthesized in Escherichia coli during various O2- -generating conditions. Nine proteins were constitutively synthesized over wild-type levels in superoxide dismutase (sodA sodB) double mutants. Addition of redox cycling agents such as paraquat and plumbagin at various concentrations induced up to 13 proteins in wild-type cells. Among these 13 were 5 of the 9 constitutively synthesized in the sodA sodB double mutants. Addition of these agents to the superoxide dismutase mutants in low micromolar concentrations induced an additional set of 14 proteins. The proteins induced included only five proteins that have been previously associated with stress responses, consisting of endonuclease IV (Nfo), three oxyR-regulated proteins, and one heat shock protein. O2- -mediated induction of the superoxide inducible (Soi) proteins in the wild type was independent of the oxyR+ gene for all but the three oxyR-regulated proteins. Analyses of proteins from three soi::lacZ gene fusions previously isolated (T. Kogoma, S. B. Farr, K. M. Joyce, and D. O. Natvig, Proc. Natl. Acad. Sci. USA 85:4799-4803, 1988) indicated the specific loss of one of these induced proteins in each fusion strain and the constitutive expression of some Soi proteins.
Autocide AMI rescues development in dsg mutants of Myxococcus xanthus
Authors: Rosenbluh A , Rosenberg E
Published: November 2016

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.
Organization of the flaFG gene cluster and identification of two additional genes involved in flagellum biogenesis in Caulobacter crescentus
Authors: Schoenlein P V , Gallman L S , Ely B
Published: November 2016

In Caulobacter crescentus, mutations have been isolated in more than 30 flagellar genes (fla, flb, and flg) which are required in the cell cycle event of flagellum biogenesis. The flaF and flaG mutations and two newly identified mutations, flbT and flbA (P.V. Schoenlein and B. Ely, J. Bacteriol. 171:000-000, 1989), have been localized to the flaFG region. In this study, the genetic and physical organization of this region was analyzed, using the cloned 4.0-kilobase flaFG region in the recombinant plasmid pPLG727. Plasmid pPLG727 complemented flaF, flaG, flbA, and flbT mutations. Further complementation studies with pPLG727 derivatives indicated that flaF and flbT are unique but overlapping transcription units, whereas flbA and flaG constitute a single transcription unit. To determine the direction of transcription of the putative flbA-flaG operon, the promoterless chloramphenicol transacetylase gene was inserted into various positions in the flbA-flaG region, and merodiploid strains containing these transcriptional fusions were assayed for gene function and expression of chloramphenicol resistance. These studies showed that transcription proceeds from flbA to flaG. To confirm the complementation analysis, Southern analyses were performed on chromosomal DNAs isolated from strains containing insertion and deletion mutations. Taken together, these studies defined the relative gene order at one end of the flaYG flagellar gene cluser as flgL-flaF-flbT-flbA-flaG.
Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures
Authors: Lipinska B , Fayet O , Baird L , Georgopoulos C
Published: November 2016

We identified and cloned an Escherichia coli gene called htrA (high temperature requirement). The htrA gene was originally discovered because mini-Tn10 transposon insertions in it allowed E. coli growth at 30 degrees C but prevented growth at elevated temperatures (above 42 degrees C). The htrA insertion mutants underwent a block in macromolecular synthesis and eventually lysed at the nonpermissive temperature. The htrA gene was located at approximately 3.7 min (between the fhuA and dapD loci) on the genetic map of E. coli and between 180 and 187.5 kilobases on the physical map. It coded for an unstable, 51-kilodalton protein which was processed by removal of an amino-terminal fragment, resulting in a stable, 48-kilodalton protein.
A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane
Authors: Winans S C , Kerstetter R A , Ward J E , Nester E W
Published: November 2016

The VirA protein is one of two proteins required for transcriptional activation of Agrobacterium tumefaciens virulence genes in response to phenolic compounds released by plants during infection. We describe two experimental approaches which indicate that this protein has a transmembrane topology. First, spheroplasts of Escherichia coli or wild-type A. tumefaciens expressing the VirA protein were treated with proteinase K to digest periplasmic proteins, and the remaining proteins were immunologically stained on Western blots (immunoblots) by using anti-VirA antibody. Second, transposon TnphoA was used to generate translational fusions between virA and phoA, the latter of which is the structural gene for alkaline phosphatase. Both techniques indicated that VirA spans the cytoplasmic membrane, with approximately 275 amino acids near the amino terminus being localized in the periplasmic space and the rest of the protein being localized in the cytoplasm. We also show that overexpression of VirA in E. coli is deleterious to cell growth and that this phenomenon depends on the synthesis of either the second hydrophobic core or some nearby portion of the VirA protein.