bluuebunny/crossref_metadata_2025 huggingface dataset:
Turnover and recycling of the murein sacculus in oligopeptide permease-negative strains of Escherichia coli: indirect evidence for an alternative permease system and for a monolayered sacculus
Authors: Park J T
Published: November 2016

Turnover of murein in oligopeptide permease-negative Escherichia coli cells appeared to be minimal or nonexistent. In one strain in which it was possible to measure turnover during the first generation of chase, it was found that the rate of turnover was constant throughout a chase of three generations. This result suggests that an "inside-to-outside" mode of growth of the sacculus does not occur in E. coli. Turnover, though minimal, was significantly higher from cells labeled uniformly than from cells labeled only in the lateral wall, suggesting that a significant portion of the observed turnover is related to cell separation. Actually, turnover only appeared to be minimal in opp mutant strains. Tripeptides were being released by turnover at a rate of about 50% per generation and then were efficiently recycled. This suggests that in addition to opp, a low-affinity uptake system for tripeptide derived from the sacculus may exist.
Rhs elements of Escherichia coli K-12: complex composites of shared and unique components that have different evolutionary histories
Authors: Zhao S , Sandt C H , Feulner G , Vlazny D A , Gray J A , Hill C W
Published: November 2016

The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined. These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements. H-rpt is found in RhsB, RhsC, and RhsE. Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E. coli genome is relatively recent. In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element. The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1%. They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF). RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted. Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected. A 291-bp fragment of H-rpt is found near min 5 of the E. coli K-12 map and is not associated with any Rhs core homology. The complete core sequences of RhsB and RhsC have been compared with that of RhsA. As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each. Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base. In each case, the core ORF extends beyond the core into the unique sequence. Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9%. All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA. A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed. RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs. One of these ORFs includes a signal peptide that is functional when fused to TnphoA.
Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area
Authors: Schneider H , Fsihi H , Kottwitz B , Mygind B , Bremer E
Published: November 2016

The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6. To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred. With one exception, these mutant Tsx proteins could still serve as a colicin K receptor. DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication. In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease. The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function. HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane.
Sequence-based differentiation of strains in the Mycobacterium avium complex
Authors: Frothingham R , Wilson K H
Published: November 2016

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.
Cloning, expression, and characterization of the icd gene in the immI operon of bacteriophage P1
Authors: Riedel H D , Heinrich J , Schuster H
Published: November 2016

The immI operon of P1 contains the genes c4, icd (formerly called orfx), and ant which are constitutively transcribed in that order from a single promoter, P51b. C4 is an antisense RNA which is processed from the precursor transcript. C4 RNA acts as a translational repressor of icd, thereby also inhibiting antirepressor (ant) synthesis. We have cloned the icd and the overlapping icd and ant genes. We show, by means of plasmid deletion analysis, that icd is translationally coupled to ant. An internal in-frame deletion of icd making up 65% of the codons still allows antirepressor synthesis at a reduced rate, indicating that a functionally active icd gene product is dispensable for ant expression. We identify the product of the icd gene as a 7.3-kDa protein which interferes with cell division. The results suggest that constitutive expression of icd, in the absence of a functionally active antirepressor, prevents P1 lysogen formation because of its detrimental effect on the host cell.
Cloning and sequencing of the low-affinity penicillin-binding protein 3r-encoding gene of Enterococcus hirae S185: modular design and structural organization of the protein
Authors: Piras G , Raze D , el Kharroubi A , Hastir D , Englebert S , Coyette J , Ghuysen J M
Published: November 2016

The clinical isolate Enterococcus hirae S185 has a peculiar mode of resistance to penicillin in that it possesses two low-affinity penicillin-binding proteins (PBPs): the 71-kDa PBP5, also found in other enterococci, and the 77-kDa PBP3r. The two PBPs have the same low affinity for the drug and are immunochemically related to each other. The PBP3r-encoding gene has been cloned and sequenced, and the derived amino acid sequence has been compared by computer-assisted hydrophobic cluster analysis with that of the low-affinity PBP5 of E. hirae R40, the low-affinity PBP2' of Staphylococcus aureus, and the PBP2 of Escherichia coli used as the standard of reference of the high-M(r) PBPs of class B. On the basis of the shapes, sizes, and distributions of the hydrophobic and nonhydrophobic clusters along the sequences and the linear amino acid alignments derived from this analysis, the dyad PBP3r-PBP5 has an identity index of 78.5%, the triad PBP3r-PBP5-PBP2' has an identity index of 29%, and the tetrad PBP3r-PBP5-PBP2'-PBP2 (of E. coli) has an identity index of 13%. In spite of this divergence, the low-affinity PBPs are of identical modular design and possess the nine amino acid groupings (boxes) typical of the N-terminal and C-terminal domains of the high-M(r) PBPs of class B. At variance with the latter PBPs, however, the low-affinity PBPs have an additional approximately 110-amino-acid polypeptide stretch that is inserted between the amino end of the N-terminal domain and the carboxy end of the membrane anchor. While the enterococcal PBP5 gene is chromosome borne, the PBP3r gene appears to be physically linked to the erm gene, which confers resistance to erythromycin and is known to be plasmid borne in almost all the Streptococcus spp. examined.
Analysis of a genomic DNA region from the cyanobacterium Synechococcus sp. strain PCC7942 involved in carboxysome assembly and function
Authors: Price G D , Howitt S M , Harrison K , Badger M R
Published: November 2016

We report on the sequencing and analysis of a 3,557-bp genomic DNA clone that is located between 4.8 and 1.2 kilobase pairs (kb) upstream of the rbcL gene and is capable of complementing a class of cyanobacterium Synechococcus sp. strain PCC7942 mutants requiring a high level of CO2. The upstream 2,704 bp of this sequence is novel, the remaining 852 bp having been reported by other workers. Four new open reading frames (ORFs) have been identified along with putative promoter elements. These ORFs, which could code for proteins of 7, 10.9, 11, and 58 kDa in size, have been named ORF 64, ccmK, ccmL, and ccmM, respectively. The last three have been named ccm genes on the basis that insertional mutagenesis of each produces a phenotype requiring a high level of CO2 (i.e., each produces a lesion in the CO2 concentrating mechanism). The putative gene product for the large ccmM ORF has three internally repeated regions and also has two possible DNA binding motifs. Two defined mutants in the 3,557-bp region, mutants PVU and P-N, have been more fully characterized. The PVU mutant has a drug marker inserted into the ccmL gene, and it possesses abnormal rod-shaped carboxysomes. The P-N mutant is a 2.64-kb deletion of DNA from the same position in ccmL to a region closer to rbcL. This mutant, which has previously been shown to lack carboxysomes and have soluble ribulosebiphosphate carboxylase/oxygenase activity, has now been shown to have a predominantly soluble carboxysomal carbonic anhydrase activity. Both mutants were found to possess carboxysomal carbonic anhydrase activities which are below wild-type levels, and in the P-N mutant this activity appears to be unstable. The results are discussed in terms of the possible interactions of putative ccm gene products in the process of carboxysome assembly and function.
Identification of an early-stage gene of Chlamydia psittaci 6BC
Authors: Wichlan D G , Hatch T P
Published: November 2016

Chlamydiae are parasitic bacteria characterized by a temporally regulated developmental cycle. In the early stage of the cycle, metabolically inert elementary bodies reorganize to dividing reticulate bodies, a process about which little is known. The purpose of this investigation was to identify and clone chlamydial genes that are expressed preferentially during the early stage of the developmental cycle of Chlamydia psittaci 6BC. Several potential early genes were cloned with highly radioactive, host-free-generated RNA probes to screen a genomic library. One clone appeared to encode a gene that was particularly well expressed at 1 h postinfection. In further characterization, we found that it encodes two complete open reading frames and one partial open reading frame of 370 codons. The partial open reading frame, designated gltX, is very similar to bacterial glutamyl-tRNA synthetases and was demonstrated to be transcribed in vivo at 24 h postinfection by primer extension analysis. A lysine-rich open reading frame (LRO) of 117 codons was found upstream and divergent from gltX. The LRO lacks homology to known proteins, and we were unable to demonstrate that it is transcribed in vivo. The third open reading frame, of 182 codons, was found to be convergent with and partially overlap the LRO. It was confirmed to be preferentially expressed within the first 1.5 h of infection by Northern (RNA) blot analysis and was designated the early upstream open reading frame (EUO). Like the LRO, the EUO is not homologous to known proteins. A major potential transcription start site of the EUO was identified by primer extension analysis. However, the sequence upstream of the site does not closely resemble the consensus recognition sequences of bacterial sigma factors even though it is AT rich. The EUO is the first chlamydial gene specific to the early stage to be cloned and sequenced.
A bifunctional enzyme, with separate xylanase and beta(1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens
Authors: Flint H J , Martin J , McPherson C A , Daniel A S , Zhang J X
Published: November 2016

Adjacent regions of a Ruminococcus flavefaciens 17 DNA fragment were found to encode xylanase and beta(1,3-1,4)-glucanase activities. Sequencing of this fragment showed that both activities are encoded by a single 2,406-bp open reading frame corresponding to the xynD gene. The predicted product has a characteristic signal sequence that is followed by an amino-terminal domain related to family G xylanases, while the carboxyterminal domain is related to beta(1,3-1,4)-glucanases from several other bacterial species. These two domains are connected by a region of unknown function that consists of 309 amino acids and includes a 30-amino-acid threonine-rich sequence. A polypeptide having a molecular weight of approximately 90,000 and exhibiting xylanase and beta(1,3-1,4)-glucanase activities was detected in Escherichia coli cells carrying the cloned xynD gene. This is one of the first cases in which a microbial polysaccharidase has been shown to carry separate catalytic domains active against different plant cell wall polysaccharides within the same polypeptide. xynD is one of a family of related genes in R. flavefaciens that encode enzymes having multiple catalytic domains, and the amino terminus of XYLD exhibits a high degree of similarity with the corresponding regions of another xylanase, XYLA, which carries two different xylanase catalytic domains.
Dienelactone hydrolase from Pseudomonas cepacia
Authors: Schlömann M , Ngai K L , Ornston L N , Knackmuss H J
Published: November 2016

Dienelactone hydrolases have previously been shown to play a crucial role in chlorocatechol degradation via the modified ortho cleavage pathway. Recently, the enzymes induced in 4-fluorobenzoate-utilizing bacteria have been classified into three groups on the basis of their specificity towards cis- and trans-dienelactone. The dienelactone hydrolase and the 3-oxoadipate enol-lactone hydrolase from Pseudomonas cepacia have now been purified to apparent homogeneity and characterized with respect to molecular mass and amino acid composition. The dienelactone hydrolase has a distinct preference for cis-dienelactone and did not convert the trans isomer or muconolactone, 3-oxoadipate enol-lactone, or 4-fluoromuconolactone to a significant extent. In properties like amino acid composition, pH optimum of activity, and lack of inhibition by p-chloromercuribenzoate, the P. cepacia dienelactone hydrolase differed substantially from 3-oxoadipate enol-lactone hydrolases and other dienelactone hydrolases.
Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1
Authors: Blatch G L , Woods D R
Published: November 2016

The Bacteroides fragilis BF-1 fructanase-encoding gene (fruA) was cloned and expressed in Escherichia coli from the recombinant plasmid pBS100. The fruA gene consisted of 1,866 bp encoding a protein of 622 amino acids with a calculated M(r) of 70,286. The apparent M(r) of the fructanase, determined by in vitro cell-free transcription-translation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, was approximately 71,500. An alignment of the amino acid sequences of the B. fragilis BF-1 fructanase and the Bacillus subtilis levanase revealed that 45.5% of the amino acids were identical. The fruA gene was expressed in E. coli from its own promoter; however, no E. coli promoter-like sequence was evident upstream from the gene. A major E. coli transcription start point and a single B. fragilis BF-1 transcription start point were located. Expression of the fruA gene was constitutive in E. coli(pBS100) and B. fragilis BF-1. The ratio of sucrase activity to inulinase activity (S/I ratio) was constant for enzyme preparations from E. coli (pBS100), indicating that both activities were associated with the fructanase. For B. fragilis BF-1, the S/I ratio varied considerably depending on the carbon source used for growth, suggesting that a separate sucrase is produced in addition to the fructanase in B. fragilis BF-1. Localization experiments and TnphoA mutagenesis indicated that the fructanase was exported to the periplasm. Sequence analysis of the N-terminal region of the fructanase revealed a putative 30-amino-acid signal peptide. The enzymatic properties of the purified fructanase were investigated. The enzyme was able to hydrolyze sucrose, raffinose, inulin, and levan but not melezitose, indicating that it was a beta-D-fructofuranosidase which was able to hydrolyze beta(2-->6)-linked fructans.
Excisive recombination of the SLP1 element in Streptomyces lividans is mediated by Int and enhanced by Xis
Authors: Brasch M A , Cohen S N
Published: November 2016

The functions mediating site-specific recombination of the SLP1 element have been mapped to a 2.2-kb region that includes the site of integration (attP), a gene (int) that specifies a function both necessary and sufficient for integration of SLP1, and an open reading frame, orf61, suspected of encoding a protein, Xis, that shows limited similarity to the excisionases of other site-specific recombination systems. Here we describe experiments that investigate the respective roles of orf61 and int in the excision of SLP1. We constructed derivatives of the high-copy-number Streptomyces plasmid pIJ101 that express orf61, int, or both orf61 and int from transcriptional fusions to the Tn5 aph gene and tested the ability of these constructs to promote excision of an adventitious attP-containing plasmid that had been integrated site-specifically into the attB site of the Streptomyces lividans chromosome. Expression of the int gene product alone from an exogenous promoter was sufficient for excision of the integrated plasmid. This result indicates that the SLP1 int-encoded protein can carry out excisive, as well as integrative, recombination. The orf61 gene product, when expressed from an exogenous promoter, inhibited int-mediated integration at the chromosomal attB site. Moreover, under conditions in which excision and transfer normally occur, precise excision of SLP1 was enhanced by the orf61-encoded protein. On the basis of these findings, we here designate the orf61 gene as xis.
The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein
Authors: Sprenger W W , Hoff W D , Armitage J P , Hellingwerf K J
Published: November 2016

The motile, alkalophilic, and extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila is positively photophobotactic. This response results in the accumulation of bacteria in light spots (E. Hustede, M. Liebergesell, and H. G. Schlegel, Photochem. Photobiol. 50:809-815, 1989; D. E. McRee, J. A. Tainer, T. E. Meyer, J. Van Beeumen, M. A. Cusanovich, and E. D. Getzoff, Proc. Natl. Acad. Sci. USA 86:6533-6537, 1989; also, this work). In this study, we demonstrated that E. halophila is also negatively phototactic. Video analysis of free-swimming bacteria and the formation of cell distribution patterns as a result of light-color boundaries in an anaerobic suspension of cells revealed the existence of a repellent response toward intense (but nondamaging) blue light. In the presence of saturating background photosynthetic light, an increase in the intensity of blue light induced directional switches, whereas a decrease in intense blue light gave rise to suppression of these reversals. To our knowledge, this is the first report of a true repellent response to light in a free-swimming eubacterium, since the blue light response in Escherichia coli and Salmonella typhimurium (B. L. Taylor and D. E. Koshland, Jr., J. Bacteriol. 123:557-569, 1975), which requires an extremely high light intensity, is unlikely to be a sensory process. The wavelength dependence of this negative photoresponse was determined with narrow band pass interference filters. It showed similarity to the absorption spectrum of the photoactive yellow protein from E. halophila.
Immunochemistry and localization of the enzyme disaggregatase in Methanosarcina mazei
Authors: de Macario E C , Macario A J , Mok T , Beveridge T J
Published: November 2016

The enzyme disaggregatase (Dag) from Methanosarcina mazei was studied immunochemically. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Dag under reducing and nonreducing conditions revealed a single band with a 94-kDa molecular mass. Dag was found to be immunogenic in rabbits; a polyclonal antibody probe was prepared and used to detect the enzyme by slide immunoenzymatic assay, immunofluorescence, and immunoblotting in various species of Methanosarcina known to convert from packets to single cells, including M. mazei. The enzyme could not be detected in other members of the family Methanosarcinaceae that do not convert. By immunogold electron microscopy, Dag was mapped to the cell wall of packets and to the cell membrane of single cells of two M. mazei strains.
Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens
Authors: Dreyfus G , Williams A W , Kawagishi I , Macnab R M
Published: November 2016

FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage. FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A. P. Volger, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). It is even more similar to the Spa47 protein of Shigella flexneri (M. M. Venkatesan, J. M. Buysse, and E. V. Oaks, J. Bacteriol. 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S. Fenselau, I. Balbo, and U. Bonas, Mol. Plant-Microbe Interact. 5:390-396, 1992), which are believed to play a role in the export of virulence proteins. Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase. FliI was overproduced and purified almost to homogeneity. It demonstrated ATP binding but not hydrolysis. An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell. An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related. The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.
Amplification of the Bacillus subtilis maf gene results in arrested septum formation
Authors: Butler Y X , Abhayawardhane Y , Stewart G C
Published: November 2016

The Bacillus subtilis homolog of the Escherichia coli morphogene orfE (of the mre operon) has been identified. The determinant is located on the chromosome immediately upstream of the mreBCD-minCD (divIVB) operon. The Maf protein shares substantial amino acid sequence identity with the E. coli OrfE protein. Introduction of the B. subtilis maf determinant on a multicopy plasmid into B. subtilis cells results in an inhibition of septation, which leads to extensive filamentation and loss of viability in the transformed cell population. Insertional inactivation of maf indicated that this gene is not essential for cell division.
Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry
Authors: Woldringh C L , Huls P G , Vischer N O
Published: November 2016

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.
ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA
Authors: Roggiani M , Dubnau D
Published: November 2016

ComA is a response regulator protein of Bacillus subtilis which is required for the transcription of several genes which are involved in late-growth expression and in responses to environmental stress. Among these genes are degQ, gsiA, and srfA. The last is an operon needed for the development of genetic competence, surfactin production, and normal sporulation. We show here that partially purified ComA protein, isolated from an overproducing Escherichia coli strain, is phosphorylated in vitro by incubation with acetyl phosphate and that ComA could bind specifically to a DNA fragment containing the promoter of srfA and associated sequences. The binding affinity is enhanced when ComA is phosphorylated. DNase I protection analysis identified two protected sites located upstream from the srfA promoter. The presence of DNase I-hypersensitive bonds induced by ComA binding which are located between the protected sequences is consistent with a model for ComA action involving the bending of DNA.
Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB
Authors: Furukawa H , Tsay J T , Jackowski S , Takamura Y , Rock C O
Published: November 2016

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the beta-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.
Regulation of glycerol metabolism in Enterococcus faecalis by phosphoenolpyruvate-dependent phosphorylation of glycerol kinase catalyzed by enzyme I and HPr of the phosphotransferase system
Authors: Deutscher J , Bauer B , Sauerwald H
Published: November 2016

Using a polyclonal antibody against glycerol kinase from Enterococcus faecalis, we could demonstrate that glycerol kinase is inducible by growth on glycerol-containing medium and that during growth on glycerol the enzyme is mainly phosphorylated. Glucose and other sugars metabolized via the Embden-Meyerhof pathway strongly repressed the synthesis of glycerol kinase, while if glycerol was also present during growth, low activity, reflecting partial induction and the presence of mainly unphosphorylated, less active enzyme, was found. With gluconate, which is also a substrate of the phosphotransferase system, repression of glycerol kinase was less severe, but the enzyme was mainly present in the less active, unphosphorylated form. Effects of growth on different carbon sources on glycerol uptake are also reported.
Methanol oxidation genes in the marine methanotroph Methylomonas sp. strain A4
Authors: Waechter-Brulla D , DiSpirito A A , Chistoserdova L V , Lidstrom M E
Published: November 2016

Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp. strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits. A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp. strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase. Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment. In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG). The moxG gene product has been shown to be cytochrome c552. The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown. Our data suggest that the order of these genes in Methylomonas sp. strain A4 is moxFJGIR, the same as in the facultative methylotrophs. The transcriptional start site for moxF was mapped. The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs. These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially.
Copy number of the 16S rRNA gene in Rickettsia prowazekii
Authors: Pang H , Winkler H H
Published: November 2016

The obligate intracellular parasite, Rickettsia prowazekii, is a slowly growing bacterium with a doubling time of 8 to 12 h. The copy number of the 16S rRNA gene in the rickettsial chromosome was determined to be one. Genomic DNA from R. prowazekii was digested either by a variety of restriction enzymes known not to cut at any site in the rickettsial 16S rRNA gene or by a combination of these noncutting enzymes and SmaI, which cuts the gene only once. Only one DNA fragment in these digests hybridized to a biotinylated probe containing a portion of the rickettsial 16S rRNA gene. Moreover, the density of the rickettsial 16S rRNA gene fragment after hybridization was equal to the density of each of the seven 16S rRNA gene fragments in Escherichia coli.
Regulatory interactions between RepA, an essential replication protein, and the DNA repeats of RepFIB from plasmid P307
Authors: Spiers A J , Bhana N , Bergquist P L
Published: November 2016

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene (BCDD'D" and EFGHIJ). In vivo genetic tests demonstrate that the BCDD'D" repeats form part of the origin of replication, while some of the downstream repeat elements (HIJ) are involved in the sensing and setting of plasmid copy number. RepA DNA binding to these groups of repeats has been investigated in vivo by utilizing the fact that the replicon contains three active promoters (orip, repAp, and EFp), one of which has previously been shown to control the expression of repA (repAp). All three promoters are closely associated with the repeat elements flanking repA, and an investigation using lacZ or cml gene fusions has demonstrated that RepA expressed in trans is able to repress each promoter. However, these assays suggest that the transcriptional responses of orip and repAp to RepA repression are significantly different, despite the fact that both promoters are embedded within the BCDD'D" repeat elements. Extra copies of the BCDD'D" or EFG repeats in trans have no effect on RepA repression of repAp embedded in a second copy of the BCDD'D" repeats, but copies of the HIJ or EFGHIJ repeats are able to derepress repAp, suggesting that there is a fundamental difference between RepA-BCDD'D" or -HIJ complexes and RepA-EFG or -EFGHIJ complexes.
RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism
Authors: Brantl S , Birch-Hirschfeld E , Behnke D
Published: November 2016

Expression of the rate-limiting initiator protein RepR of plasmid pIP501 is controlled by the antisense RNAIII. Mutational alteration of individual G residues within the single-stranded loops of RNAIII led to an increase in copy number. In contrast to the G-rich single-stranded loops, two smaller AT-rich loops of RNAIII were found to be dispensable for its inhibitory function. Reciprocal mutations in the same loop compensated for each other's effect, and a destabilization of the major stem structure of RNAIII also resulted in an increased copy number. These data were consistent with the idea that the interaction of RNAIII with its target starts with the formation of a kissing complex between the single-stranded loops of both molecules. The repR mRNA leader sequence, which includes the target of RNAIII, is able to assume two alternative structures due to the presence of two inverted repeats the individual sequences of which are mutually complementary. In the presence of the antisense RNAIII, one of these inverted repeats (IR2) is forced to fold into a transcriptional terminator structure that prevents transcription of the repR gene. In the absence of RNAIII, formation of the transcriptional terminator is prevented and expression of the essential repR gene can proceed normally. This antisense RNA-driven transcriptional attenuation mechanism was supported by extensive deletional analysis and direct evidence that IR2 functions as a transcriptional terminator.
High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature
Authors: Nemecek-Marshall M , LaDuca R , Fall R
Published: November 2016

Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C). Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h. The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression. Pulse-labeling of nongrowing cultures with [35S]methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced. Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h. Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells. Not all P. syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature. In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R. L. Green and G. Warren, Nature [London] 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M. Vincente, S. R. Kushner, T. Garrido, and M. Aldea, Mol. Microbiol. 5:2085-2091, 1991).
Gene structure and expression of the Corynebacterium flavum N13 ask-asd operon
Authors: Follettie M T , Peoples O P , Agoropoulou C , Sinskey A J
Published: November 2016

Two promoters required for expression of the ask-asd genes, encoding aspartokinase (AK) and aspartate-semialdehyde dehydrogenase (ASD), in Corynebacterium flavum N13, askP1 and askP2, have been identified by deletion analysis and S1 nuclease mapping. Transcription from askP1 initiates 35 and 38 bp upstream of the ask structural gene. A second promoter, askP2, lies within the ask coding region, upstream of the translation start site of the AK beta subunit and can direct the expression of AK beta and ASD. Western immunoblot analysis and heterologous expression in Escherichia coli demonstrate that two separate polypeptides, a 44.8-kDa alpha subunit and an 18.5-kDa beta subunit, are expressed from the C. flavum N13 ask gene from distinct, in-frame translation initiation sites. A second AK mutation, G345D, which reduces the sensitivity of AK to concerted feedback inhibition by threonine plus lysine, was identified.
Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction
Authors: Wall J D , Rapp-Giles B J , Rousset M
Published: November 2016

A 2.3-kb plasmid present in about 20 copies per genome was identified in extracts of Desulfovibrio desulfuricans G100A and designated pBG1. It appears to be unable to replicate in Escherichia coli. Although composite plasmids of pBG1 inserted into pTZ18U are stable in E. coli, few if any pBG1-specific transcripts are detectable. The plasmid sequence reveals several features typical of the origin of replication of non-ColE1 enterobacterial plasmids as well as several potential open reading frames. This small replicon has been shown to support the replication of recombinant plasmids in D. desulfuricans G100A and Desulfovibrio fructosovorans. A conjugable shuttle vector has been constructed.
Diversity of retron elements in a population of rhizobia and other gram-negative bacteria
Authors: Rice S A , Bieber J , Chun J Y , Stacey G , Lampson B C
Published: November 2016

Genetic elements called retrons reside on the chromosome of Escherichia coli and the myxobacteria and represent the first reverse transcriptase-encoding element to be found in a prokaryotic cell. All known retrons produce a functionally obscure RNA-DNA satellite molecule called multicopy single-stranded DNA (msDNA). We report here the presence of msDNA-producing retron elements in a number of new bacterial groups, including strains of the genera Proteus, Klebsiella, Salmonella, Nannocystis, Rhizobium, and Bradyrhizobium. Among a population of 63 rhizobia strains, only 16% contain a retron element. The rhizobia retrons appear to be heterogeneous in nucleotide sequence and show little similarity to previously studied retrons of E. coli and the myxobacteria.
Activation of the Bacillus subtilis hut operon at the onset of stationary growth phase in nutrient sporulation medium results primarily from the relief of amino acid repression of histidine transport
Authors: Atkinson M R , Wray L V , Fisher S H
Published: November 2016

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.
Physical mapping of stable RNA genes in Bacillus subtilis using polymerase chain reaction amplification from a yeast artificial chromosome library
Authors: Okamoto K , Serror P , Azevedo V , Vold B
Published: November 2016

A new approach for mapping genes which utilizes yeast artificial chromosome clones carrying parts of the Bacillus subtilis genome and the polymerase chain reaction technique is described. This approach was used to physically map stable RNA genes of B. subtilis. Results from over 400 polymerase chain reactions carried out with the yeast artificial chromosome clone library, using primers specific for the genes of interest and designed from published sequences, were collected. The locations of 10 known rRNA gene regions (rrnO, rrnA, rrnE, rrnD, rrnB, rrnJ-rrnW, and rrnI-rrnH-rrnG) have been determined by this method, and these results correlate with those observed by standard genetic mapping. All rRNA operons, except rrnB, are found between 0 and 90 degrees, while rrnB has been placed in the area of 270 degrees on the chromosome map. Also localized were the tRNA gene clusters associated with the following ribosomal operons: rrnB (21 tRNAs), rrnJ (9 tRNAs), rrnD (16 tRNAs), and rrnO and rrnA (2 internal tRNAs). A previously unmapped four-tRNA gene cluster, trnY, a tRNA gene region that is not associated with a ribosomal operon, was found near the origin of replication. The P-RNA gene, important for processing of tRNAs, was found between map locations 197 and 204 degrees.
Characterization of a cytochrome a1 that functions as a ubiquinol oxidase in Acetobacter aceti
Authors: Fukaya M , Tayama K , Tamaki T , Ebisuya H , Okumura H , Kawamura Y , Horinouchi S , Beppu T
Published: November 2016

The terminal oxidase for ethanol oxidation in Acetobacter aceti was purified as a complex consisting of four subunits (subunits I, II, III, and IV) with molecular masses of 72, 34, 21, and 13 kDa, respectively. Spectrophotometric analysis and catalytic properties determined with the purified enzyme showed that it belonged to a family of cytochrome a1 (ba)-type ubiquinol oxidases. A polymerase chain reaction with two oligonucleotides designed for amino acid sequences that are conserved in subunit I of the aa3-type cytochrome c oxidases from various origins and of an Escherichia coli o (bo)-type ubiquinol oxidase was used for cloning the cytochrome a1 gene. A 0.5-kb fragment thus amplified was used as the probe to clone a 4.5-kb KpnI fragment that contained a putative open reading frame for the whole subunit I gene. The molecular weight and amino acid composition of the product of this open reading frame (cyaA) were the same as those of the purified protein from A. aceti. The amino acid sequence of CyaA was homologous to that of subunit I of the E. coli o-type ubiquinol oxidase. Nucleotide sequence analysis of the region neighboring the cyaA gene revealed that the genes (cyaB, cyaC, and cyaD) encoding the other three subunits (subunits II, III, and IV) were clustered upstream and downstream of the cyaA gene in the order cyaB, cyaA, cyaC, and cyaD and with the same transcription polarity, forming an operon. As expected from the enzymatic properties, CyaB, CyaC, and CyaD showed great similarity in amino acid sequence to the corresponding sununits of the E. coli o-type ubiquinol oxidase and as(3)-type cytochrome c oxidases.