scholarly journals Purification of a class C A-type β-lactamase from a derepressed strain of Enterobacter cloacae. Comparison of the wild-type and mutant enzyme with those from strains P99, 208 and GN7471

1989 ◽  
Vol 260 (3) ◽  
pp. 705-710 ◽  
Author(s):  
M N Graham ◽  
T J Mantle
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Enterobacter Cloacae ◽  
Cross Reactivity ◽  
Permeability Barrier ◽  
Beta Lactamase ◽  
Wild Type ◽  
Beta Lactams ◽  
Class C

Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. ‘good’ substrates with kcat. values of 80-1200 s-1 and ‘poor’ substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.

scholarly journals Sequencing and analysis of four new Enterobacter ampD Alleles.

1996 ◽  
Vol 40 (8) ◽  
pp. 1953-1956 ◽  
Author(s):  
A F Ehrhardt ◽  
C C Sanders ◽  
J R Romero ◽  
J S Leser
Keyword(s):  
Amino Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Enterobacter Cloacae ◽  
Temperature Sensitive ◽  
Beta Lactamase ◽  
Wild Type ◽  
The Family

Sequences of ampD genes from wild-type, temperature-sensitive, and stably derepressed mutants of the wild-type strain of Enterobacter cloacae 029 and the hyperinducible strain E. cloacae 1194E were determined and compared with the ampD gene of the wild-type strain E. cloacae 14. Seventy nucleotide differences were found between the wild-type sequences, resulting in 13 amino acid changes. The deduced amino acid changes do not correspond to published AmpC regulation mutations and expand the number of known mutations leading to altered AmpC beta-lactamase expression in members of the family Enterobacteriaceae.

scholarly journals Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

2004 ◽  
Vol 186 (5) ◽  
pp. 1374-1380 ◽  
Author(s):  
Ayako Furutani ◽  
Seiji Tsuge ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Secretion System ◽  
Xanthomonas Oryzae ◽  
Nucleotide Sequence Analysis ◽  
Secretory Proteins ◽  
Type Ii ◽  
Wild Type ◽  
Type Iii

ABSTRACT Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

scholarly journals Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways

2002 ◽  
Vol 184 (23) ◽  
pp. 6559-6565 ◽  
Author(s):  
Alvaro de la Fuente ◽  
Luis M. Lorenzana ◽  
Juan F. Martín ◽  
Paloma Liras
Keyword(s):  
Clavulanic Acid ◽  
Type Strain ◽  
High Performance ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Bioactive Compound ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Reading Frame ◽  
Acid Pathway

ABSTRACT A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a β-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wei Liu ◽  
Xue Bai ◽  
Yan Li ◽  
Haikun Zhang ◽  
Xiaoke Hu
Keyword(s):  
Signal Transduction ◽  
Host Plant ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Response Regulators ◽  
Free Living ◽  
Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

scholarly journals Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus

2021 ◽  
Vol 9 (4) ◽  
pp. 676
Author(s):  
Ting-Yu Liu ◽  
Sheng-Hui Tsai ◽  
Jenn-Wei Chen ◽  
Yu-Ching Wang ◽  
Shiau-Ting Hu ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Opportunistic Pathogen ◽  
Mycobacterium Abscessus ◽  
Colony Morphology ◽  
Clinical Strain ◽  
Immunocompromised Patients ◽  
Clinical Infection ◽  
Wild Type ◽  
Sliding Motility

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

scholarly journals Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruiqi Wang ◽  
Kun Li ◽  
Jifang Yu ◽  
Jiaoyu Deng ◽  
Yaokai Chen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Clinical Isolates ◽  
Aminobenzoic Acid ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Expression Levels ◽  
Encoding Gene ◽  
Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals Homology-Dependent Silencing of the SC3 Gene in Schizophyllum commune

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 589-596 ◽  
Author(s):  
Theo A Schuurs ◽  
Eveline A M Schaeffer ◽  
Joseph G H Wessels
Keyword(s):  
Gene Silencing ◽  
Genomic Dna ◽  
Type Strain ◽  
Cytosine Methylation ◽  
Wild Type Strain ◽  
Schizophyllum Commune ◽  
Southern Analysis ◽  
Transcriptional Level ◽  
Wild Type

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4°, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

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